CN102231979B - Liposome efficient delivery methods and compositions for gene silencing therapeutics - Google Patents

Abstract

The present invention provides methods and compositions for liposomal delivery of therapeutics prepared by contacting an aqueous solution of an active agent with a solution of liposome forming components containing one or more DILA2 amino acid compounds or lipids in an organic solvent to form an impinging fluid. The present invention provides methods comprising controlling the formation of liposomal compositions for therapeutic applications using flow rates, pH, and incubation times. The impinging fluid may be collected and incubated to produce a liposome formulation encapsulating the active agent. The composition may be quenched with buffer and filtered by tangential flow and diafiltration, as well as other means used to form a pharmaceutical composition. The present invention provides for the effectiveness of delivering drug loads. The composition may comprise a liposome comprising one or more carrier particles, wherein each carrier particle has an active agent and a peptide, wherein the ratio of the mass of the peptide plus the mass of the liposome to the mass of the active agent is less than about 15.

Description

Liposome efficient delivery methods and compositions for gene silencing therapeutics

technical field

The present invention generally relates to methods, compositions and uses for the delivery of biologically active and pharmaceutical agents. The methods and compositions of the invention can be used to deliver therapeutic agents to selected cells, tissues, organs or subjects. Embodiments of the invention may provide for the delivery of drugs and therapeutic agents, including nucleic acid agents, and methods of making and using substances for drug delivery. In particular, the invention relates to methods and compositions comprising liposomes or lamellar vesicles, and other forms of delivery enhancing compositions and formulations, as well as methods of treatment and uses of these delivery substances.

sequence listing

This application contains a Sequence Listing submitted here via EFS-Web as an ASCII file named MD-08-16PCT.txt created October 14, 2009, 99,662 bytes in size, which is hereby incorporated by reference in its entirety.

Background technique

Delivery of a therapeutic compound to a subject can be hampered by limitations in the ability of the compound to reach target cells or tissues, or by entry or passage of the compound within the cell. Delivery of therapeutic substances is often restricted to cell membranes. These barriers and limitations to delivery may result in the need to use higher concentrations of the compound than to achieve the desired result, thus carrying the risk of toxic effects and side effects.

A further constraint in the delivery of certain therapeutic compounds is the need to protect the compound from degradation during transport. In particular, systemic delivery through the blood circulation can subject the compounds to a variety of proteins, enzymes, and immunological components and factors.

One delivery strategy utilizes natural or synthetic lipophilic or polymeric carrier molecules to improve the transport of compounds into cells. These substances can take advantage of mechanisms that exist for selective entry into cells while still excluding foreign molecules such as nucleic acids and proteins. For example, cationic lipids can interact with pharmaceutical agents and make contact with cell membranes. Certain natural and synthetic lipophilic molecules can also be organized into liposomes or particles as carriers for pharmaceutical agents. Nano- or submicron-sized liposomes can take advantage of existing mechanisms for selective entry into cells, such as endocytosis. Liposome drug carriers can protect drug molecules from degradation while improving their uptake by cells. Moreover, liposomal drug carriers can encapsulate or bind certain compounds through electrostatic and other interactions, and can interact with negatively charged cell membranes to initiate transmembrane transport.

A disadvantage of liposomes is that the biological activity of therapeutic liposomal formulations is often dependent on the degree of loading of the active agent into the liposomes. Generally, a high degree of loading is desired to provide high therapeutic activity. Furthermore, loading of liposomes requires additional carrier molecules to remain within the formulation.

Another limitation of the use of liposomes for the delivery of pharmaceutical agents is that the use of liposomes increases the mass of the delivery formulation. The biological activity of a therapeutic formulation and its relative toxicity will be affected by the nature and quality of other components used to prepare the formulation, such as lipophilic molecules and carriers. Conventional lipid-based liposome formulations for delivery of active agents can have a 10 to 15-fold higher ratio of the mass of lipid molecules to the mass of active agent. In general, lower ratios of lipid or carrier to active agent are more effective and desirable. For such liposome formulations for nucleic acid agents, each liposome may contain no higher than several hundred copies of nucleic acid agent molecules.

Among others, the understanding of regulatory RNAs and the development of RNA interference (RNAi), RNAi therapeutics, RNA pharmaceuticals, antisense therapeutics, and gene therapy have increased the need for efficient means of introducing active nucleic acid agents into cells. Typically, nucleic acids are only stable for a limited time within cells or plasma. However, nucleic acid-based agents are capable of remaining stable in compositions and formulations that can subsequently be dispersed for cellular delivery.

There is a need for methods, compositions and applications for the systemic and local delivery of drugs and biologically active molecules. Among other things, there is a need for methods for preparing and using delivery structures and vehicles, including liposomal forms, that increase the efficiency of delivery of bioactive therapeutic molecules. It would be desirable to utilize reduced amounts of carrier material, especially for liposomal formulations, gene silencing therapeutics and other formulations, resulting in efficient delivery and high biological activity.

Contents of the invention

The present invention provides novel methods, compositions and formulations for the intracellular and in vivo delivery of pharmaceutical agents that ultimately serve as therapeutic agents, generally maintaining cytoprotection and with relatively low toxicity. The methods and compositions of the invention can be used to deliver pharmaceutical agents to selected cells, tissues and organs.

In some aspects, the invention provides processes, compositions and methods for delivering active nucleic acid agents or molecules to cells. The active agent can produce a therapeutic or pharmacological effect either by drug action or by generating a response to RNA interference or antisense or ribozyme action. Agents of the invention can be used to modulate gene expression or in gene therapy.

Embodiments of the present invention provide various methods for preparing compositions including liposomal compositions containing one or more active agents by providing a first fluid, providing a second fluid comprising a non-aqueous solution of one or more liposome-forming compounds in an organic solvent, causing the first fluid to collide with the second fluid, thereby forming said organic solvent at a concentration of about 20% to About 50% v/v impinging stream. The pH of the impingement fluid may be from about 6 to about 7.4. The impingement fluid may be incubated in the collection vessel at a temperature of about 20°C to about 35°C for about 0.5 hours to about 8 hours, thereby forming a liposome-containing incubation.

In certain embodiments, the method for preparing a composition comprising one or more active agents may comprise, by adding to the incubation a sufficient concentration of the organic solvent to be less than about 20% v/v buffer to quench the incubation.

In some aspects, a liposome-forming compound of the invention can be one or more DILA2 amino acid compounds. DILA2 amino acid compounds are synthetic organic compounds containing amino acid groups that can form liposomes. DILA2 amino acid compounds may contain a delivery-enhancing tail or a lipophilic tail at the N-terminal or C-terminal or both ends of the amino acid group.

In some variations, the method for preparing a composition containing one or more active agents may further comprise causing the volume flow rate of the first fluid containing the active agent to be greater than that of the second fluid containing liposome-forming molecules. twice the volumetric flow rate or higher. In some variations, the volumetric flow rate of the first fluid is three times or greater than the volumetric flow rate of the second fluid, or five times or greater than the volumetric flow rate of the second fluid.

The active agent of the present invention may be a UsiRNA, a nucleic acid-containing agent, a gene silencing agent, a gene modulating agent, an antisense agent, a peptide nucleic acid agent, a ribozyme agent, an RNA agent or a DNA agent. In some embodiments, the active agent can be a pharmaceutical compound or a small molecule drug.

The method for preparing a liposome composition containing one or more active agents may further comprise adding a buffer to the collection vessel to adjust the concentration of the organic solvent. The pH of the impingement fluid may be adjusted to be from about 3 to about 6. The incubation step can be performed at a pH of about 3 to about 6.

In certain aspects, the active agent can be entrapped in liposomes at a level of greater than about 50%, or greater than about 70%.

In some aspects, the invention can provide liposome compositions that retain gene silencing activity for up to 7 days at a temperature of 45°C. In certain aspects, the liposome composition can maintain encapsulation of the active agent at a temperature of 45°C for up to 7 days.

The methods of the invention may comprise filtering the incubation by tangential flow filtration and diafiltration. After tangential flow filtration, the liposomes are uniform in size and may have a diameter of less than about 160 nm, or may have an average diameter of about 40 nm to about 160 nm, or about 80 nm to about 150 nm. In a further embodiment, the incubation can be sterilized and the organic solvent can be exchanged with a different pharmaceutically acceptable buffer.

In certain methods of the invention, the incubation may be filtered by tangential filtration and diafiltration. In certain embodiments, the incubation may be sterilized.

The method of the present invention may comprise adding an organic solvent to the first fluid at a concentration of about 1% to about 40% v/v.

The organic solvent can be a sterile injectable aqueous solution having a (C1-6) alkanol concentration of about 40% to about 99% v/v or about 70% to about 95%.

The length of incubation in the methods of the invention can be from about 1 hour to about 4 hours.

The invention further relates to a pharmaceutical composition prepared by any variant of the method of the invention.

In general, the invention includes methods for delivering therapeutic nucleic acids to biological cells.

In some embodiments, the invention provides methods of inhibiting gene expression in a biological cell by preparing a composition according to the methods of the invention and treating the cell with the composition.

The methods disclosed herein for inhibiting gene expression in a mammal comprise preparing a composition according to the methods of the invention and administering the composition to said mammal.

Embodiments of the present invention may further provide methods of treating human diseases, wherein the diseases are cancer, bladder cancer, liver cancer, liver disease, hypercholesterolemia, Inflammatory diseases, metabolic diseases, inflammation, arthritis, rheumatoid arthritis, encephalitis, bone fractures, heart disease and viral diseases.

The present invention further relates to the use of the composition for treating diseases and the use of the composition in the preparation of medicaments for treating diseases.

The present invention provides various compositions and formulations for the delivery of biological agents to cells. More particularly, in certain aspects, the present invention provides liposomal formulations and carrier particles that are nanoscale in size. The carrier particles can be loaded within liposomes, can exhibit increased stability in delivery, and can effectively deliver pharmaceutical agents to modulate gene expression or activity.

The present invention provides compositions, methods and uses for improved systemic and local delivery of drugs and bioactive molecules. In particular, the present application also provides novel compositions and methods for making and using delivery structures and vehicles that can deliver active agents intracellularly with increased delivery efficiency.

In some embodiments, the invention provides compositions comprising liposomes comprising one or more carrier particles each comprising an active nucleic acid agent and a peptide wherein the mass of the peptide is and the ratio of the sum of the mass of the liposome to the mass of the nucleic acid reagent is less than about 15, or less than about 12, or less than about 10, or less than about 9, or less than about 8, or less than about 5.

In certain embodiments, the present invention provides compositions having a knockdown activity of 50% or higher, or 70% or higher, or 90% or higher in vivo for ApoB gene silencing.

In some variations, the compositions of the invention may comprise liposomes containing amino acid lipids.

In certain aspects, compositions of the invention may comprise charged carrier particles, negatively charged carrier particles, or positively charged carrier particles.

In certain variations, the active nucleic acid agent may be an RNAi-inducing agent or an antisense agent. Each liposome may contain 500 or more copies, or 1000 or more copies, or 5000 or more copies of active agent molecules.

In some variations, the peptides used in the carrier particles may be cleavable peptides or cross-linkable peptides. In some embodiments, the peptide is PN4110 or PN183.

The invention provides a method for delivering an active nucleic acid agent to a cell comprising preparing a liposome composition and treating the cell with the composition.

In certain aspects, the invention provides methods for inhibiting gene expression in a cell, the method comprising preparing a liposomal composition and treating the cell with the composition.

In certain aspects, the invention provides methods for inhibiting gene expression in a mammal, the methods comprising preparing a liposomal composition and administering the composition to the mammal.

In some embodiments, the present invention provides a method of treating a human disease selected from inflammatory diseases including rheumatoid arthritis, metabolic diseases including hypercholesterolemia, liver disease, encephalitis, bone fractures, cardiac diseases, viral diseases including hepatitis and influenza, and cancer, the method comprising preparing a liposomal composition and administering the composition to a human.

In certain embodiments, the present invention provides the use of a liposomal composition in the manufacture of a medicament for the treatment of diseases including inflammatory diseases including rheumatoid arthritis, metabolic disorders including hypercholesterolemia disease, liver disease, encephalitis, bone fractures, heart disease, viral diseases including hepatitis and influenza, and cancer.

In certain variations, the present invention provides the use of liposomal compositions for the treatment of diseases including inflammatory diseases including rheumatoid arthritis, metabolic diseases including hypercholesterolemia, liver disease, Encephalitis, bone fractures, heart disease, viral diseases including hepatitis and influenza, and cancer.

The content of the invention and the detailed description of the present invention, as well as the drawings, the appended embodiments and the claims are taken as the disclosure content of the present invention as a whole.

Description of drawings

Figure 1 : Schematic representation of a liposome embodiment of the invention wherein certain liposome-forming compounds form bilayer vesicles 10 . In this embodiment, the outer layer of the liposome is protected by polyethylene glycol chains 20 attached to the headgroup of a liposome-forming molecule. The outer layer of the liposome also has ligands 30 that specifically target cells or tissues. In this embodiment, the liposomal vesicles comprise a payload of active interfering RNA components comprising condensed RNA nanoparticles 40, double-stranded RNA duplex peptide conjugates 50, triple-stranded mdRNA 60, diced Enzyme substrate RNA 70, dsRNA with long overhangs 80 and siRNA with blunt ends 90, these components are incorporated into this embodiment.

Figure 2: A flow diagram of certain embodiments for preparing liposome compositions of the invention. A reagent solution comprising an active agent solution, a buffer solution, and a solution of one or more liposome-forming compounds is separately prepared and placed in container 200 . The solution of the active agent and the solution of the liposome-forming compound are contacted in impingement fluid 210 . The impingement fluid is collected in collection container 220 . The collected material is placed in the vessel for an incubation process 230, which is followed by quenching 240 to stabilize the material. The quenched material is subjected to a filtration process 250 . The filtrate output 260 continues with the finishing process.

Figure 3: A flow diagram for formulating certain embodiments of liposome compositions of the invention. The liposome composition (which may be a filtrate output from the preparation of the liposome composition) is sterilized 300 . Vessels for carrying the sterilized composition are filled at 310 and organized at 320 before the sterile composition is frozen at 330 and stored at 340 . The final composition is shipped 350 ready for use.

Figure 4: A flow diagram of certain embodiments for preparing liposome compositions of the invention. The active agent solution is maintained in container 400 . A solution containing liposome-forming components (eg, DILA2 compound or liposomes) is maintained in a separate container 410 . The buffer solution is maintained in another container 420 . The solution is pumped using an independent peristaltic pump 430 at an independently selected flow rate. The solution can optionally be passed through an in-line filter or filtered before filling into a vessel. During impingement, the active agent solution is contacted at contact point 434 with a solution containing liposome-forming components. The impingement fluid may optionally flow through one or more mixing tubes 436 . The impingement fluid enters the collection vessel 440 for the incubation process. The quench process buffer solution may optionally be maintained in a separate container 450 . The liposome composition 460 exits the collection vessel 440 and enters the filtration process.

Figure 5: Flow diagram of certain embodiments for preparing liposome compositions of the invention. The stabilized liposome composition from output 460 of the collection container is placed in container 500 . The diafiltered buffer solution is maintained in a separate container 520 . Peristaltic pump 502 provides circulation of the stabilized liposome composition from container 500 through tube 504 containing hollow fiber membranes. Tangential flow occurs through this cycle to concentrate the stabilized liposome composition by removing the filtrate 530 . Addition of displacement buffer from container 520 may allow for diafiltration at fixed or variable volumes. Optionally, dialysis of the stabilized liposome composition can be performed using peristaltic pump 506 to drive dialysis buffer solution from container 540 . The stabilized liposome composition at a specific concentration is output 550 to a finishing process.

Figure 6: Figure 6 shows the binding of polyarginine binding domains to dsRNAs that increase the length of the polyarginine binding domains. In Figure 6, the strongest binding (with the best ability to displace the SYBR-Gold dye) was observed with PN3499 (dimeric peptide containing a total of 10 arginines).

Detailed ways

Embodiments of the invention also provide for the delivery of drugs and therapeutic agents, including nucleic acid agents, and methods of making substances and using them for drug delivery.

The present invention further relates to novel drug delivery enhancing methods and compositions useful for delivering various molecules and structures to cells. The present invention provides methods, compounds, Compositions, formulations and applications. More particularly, the invention relates to methods and compositions comprising liposomes or lamellar vesicles, and other forms of delivery enhancing compositions and formulations, and methods of treatment and uses of these delivery substances.

The methods and compositions of the invention can further be used to deliver therapeutic, prophylactic, and diagnostic agents, such as nucleic acid agents, polynucleotides, peptides, proteins, and small molecule compounds and drugs.

The compositions and methods of the invention are useful for the delivery of therapeutic agents in a form such as encapsulated in liposomes or lamellar vesicles. These forms can include nanoparticles of various diameters.

Among them, the understanding of regulatory RNA and the development of RNA interference (RNAi or iRNA), RNAi therapy, RNA drugs, antisense therapy or ribozyme therapy, and DNA gene therapy have increased the impact on effective means of introducing active nucleic acid agents into cells demand. Typically, nucleic acids are only stable for a limited time when introduced into cells or plasma. However, nucleic acid-based agents can be stabilized in compositions and formulations that can then be administered and dispersed for cellular delivery.

Nucleic acid agents include any molecule that contains nucleic acid, such as gene silencing agents, gene modulators, antisense agents, peptide nucleic acid agents, ribozyme agents, RNA agents, and DNA agents.

In some embodiments, the compositions and methods of the invention can be used to deliver therapeutic agents encapsulated in liposomes. In these embodiments, the therapeutic agent can be referred to as a payload.

For example, FIG. 1 shows a schematic diagram of a liposome embodiment of the invention in which certain lipophilic molecules form bilayer vesicles 10 . In this embodiment, the outer layer of the liposome is protected by polyethylene glycol chains 20 attached to the headgroup of a lipophilic molecule. The outer layer of the liposome also has ligands 30 that specifically target cells or tissues. In this embodiment, the liposomal vesicles contain a payload of active RNA components including condensed RNA nanoparticles 40, double-stranded RNA duplex peptide conjugates 50, triple-stranded mdRNA 60, Dicer substrate Target RNA 70, dsRNA with long overhangs 80, and siRNA with blunt ends 90, these components are incorporated into this embodiment. Other forms of therapeutic agent loading may include microRNA, hairpin RNA, DNA or ribozyme formats.

In general, any active agent can be used as a load in the methods and compositions of the invention. In some embodiments, the cargo can be a small organic molecule pharmaceutical agent. In certain embodiments, the load can be a negatively charged therapeutic agent or a neutral therapeutic agent.

The methods and compositions provided by some aspects of the invention can deliver a therapeutic agent in a releasable form or composition. Releasable forms and compositions include molecules that bind and release the active agent, molecules that bind the active agent and unload to facilitate the release of the agent's moiety, bind the active agent and then within the biological compartment to facilitate the release of the agent Modified molecules in the manner of release, and compositions comprising the active agent-bound molecules in admixture with a release mediator compound.

In certain aspects, the invention provides methods and apparatus for preparing liposomal compositions suitable for delivery of therapeutic agents. In certain embodiments, the active agent of the invention is UsiRNA. The methods of the invention can provide liposomal compositions of nucleic acid agents, such as double or triple stranded RNA constructs, RNA peptide conjugates, condensed RNA nanoparticles, Dicer substrate RNA, dsRNA, siRNA, microRNA, Hairpin RNA and other reactive RNA forms.

The active agent of the invention may be a peptide condensate of an active RNA agent. For example, nanoparticles formed by condensation of active RNA agents and peptides or other biomolecules, condensates of RNA and polymeric species can be loaded into liposome compositions of the invention. The nanoparticles may be crosslinked.

In further embodiments, the active agent of the invention may be a peptide, protein, protease, antibody, monoclonal antibody, antibody-based drug, vaccine agent, or small molecule drug.

The composition containing the active agent may be an aqueous solution.

As used herein, the term "aqueous solution" refers to an aqueous solution, a sterile aqueous solution, or any solution in which the predominant solvent is water. Aqueous solutions may contain, for example, some organic solvents.

Examples of aqueous solvents include water, sterile water for injection, Ringer's solution, and isotonic sodium chloride solution.

Compositions containing liposome-forming molecules or lipophilic molecules may be non-aqueous solutions.

As used herein, the term "non-aqueous solution" refers to any solution in which the primary solvent is other than water. Non-aqueous solutions may contain some water.

Examples of nonaqueous solvents include water, alkanol, (C1-6) alkanol, ethanol, isopropanol, isobutanol, sec-butanol, t-butanol, alkanol-water, ethanol-water, acetonitrile, Organic solvent mixed with acetone, ketone, dimethyl sulfoxide, surfactant solution, detergent solution and their mixture.

Methods for liposome compositions

Embodiments of the invention may provide methods for preparing liposome-containing compositions containing active agents and methods of drug delivery.

In some aspects, liposome compositions can be prepared by colliding two compositions, eg, a composition containing one or more active agents and a separate composition containing one or more liposome-forming molecules.

Typically, the liposomal structures of the invention are not constructed in fully active form until all steps of the manufacturing process have been performed. Each step of the method of the present invention requires a certain amount of time. In general, the formation ratio of a liposome composition will depend on the unpredictable action of a combination of variables such as flow rate, temperature, pH, and concentration of individual components. In some embodiments, the incubation time is used to control the formation process.

Figure 2 shows a flow diagram for preparing certain embodiments of liposome compositions of the invention. In FIG. 2 , a reagent solution comprising an active agent solution, a buffer solution, and a solution of one or more liposome-forming compounds is separately prepared and placed in a solution container 200 . The reagent solutions may optionally be filtered separately or prepared under sterile conditions. The solution of the active agent and the solution of the liposome-forming compound are contacted during the impingement process 210 . The impingement fluid is collected in collection container 220 . The collected material is kept in the container for the incubation process 230, which is followed by quenching 240 to stabilize the material. The quenched material is subjected to a filtration process 250 . The filtered output is removed 260 to the finishing process.

The present invention includes embodiments of methods for preparing liposome compositions that include the impingement process. The impingement process may have one or more steps of generating an impingement fluid by impacting a composition comprising an active agent with a composition comprising a liposome-forming molecule.

In some aspects, the methods of the invention for preparing liposomal compositions of active agents can have one or more steps of the incubation process. The incubation process may include collecting the impingement fluid and maintaining it in the container for the incubation time.

Embodiments of the invention may further include methods for preparing liposome compositions, wherein the incubated compositions are quenched. Quenching can be performed by adding a solvent, buffer or diluent to the incubating composition fluid or mixture of compositions. Quenching can dilute the concentration of a particular component, such as an organic solvent or dispersant, below a predetermined level. Typically, quenching of the incubated composition can result in a stable composition in further processing or finishing steps. The step of quenching is operable to stabilize the incubated composition which may comprise liposomal structures.

Incubation of the impingement fluid in the collection vessel, as well as other steps of the method, can provide a liposomal formulation in which the active agent is highly encapsulated by the liposomes. For example, in certain embodiments, the formation of liposome compositions and structures of the invention may require any of the steps of impacting, mixing, diluting, collecting, incubating, adjusting pH, quenching, and filtering.

Methods for preparing liposome compositions of the invention may further comprise one or more filtration steps. Filtration steps can be used to alter various process parameters, such as controlling or changing the concentration of components or changing particle size or dispersibility and other physical solution parameters.

Figure 3 shows a flow diagram for formulating certain embodiments of liposome compositions of the invention. Referring to FIG. 3 , the liposome composition (which may be a filtrate output from the preparation of the liposome composition) is sterilized 300 . Containers for carrying the sterile composition are filled at 310 and processed at 320 before the sterile composition is frozen at 330 and stored at 340 . The final composition is shipped 350 ready for use.

The procedures and methods known in the art, such as those described in Remington's Pharmaceutical Sciences (18th ed. 1990), may be used in the process of sterilization, filling and finishing of substances into containers and storage of the resulting formulations involved in the present invention.

Some methods for assessing the encapsulation, size and general preparation of liposomes are described, for example, in WO2001005374, US Patent Publication Nos. 20040142025 and 20070252295, and US Patent No. 6,843,942.

crash and mix

In certain aspects, the invention provides various methods and processing conditions for preparing liposomal compositions of active agents.

Figure 4 shows a process diagram for preparing certain embodiments of liposome compositions of the invention. Referring to FIG. 4 , an active agent solution is maintained in container 400 . A solution containing liposome-forming components (eg, DILA2 amino acid compound or liposomes) is maintained in a separate container 410 . The buffer solution is maintained in another container 420 . The solution is drawn through the transfer tube 402 using an independent peristaltic pump 430 at an independently selected flow rate. The solution can optionally be passed through an in-line filter or filtered before filling into the corresponding container. During impingement, the active agent solution may be contacted at contact point 434 with a solution containing liposome-forming components. The contact points can be of any shape, angle, orientation or size. The impingement fluid may optionally flow through one or more turbulent mixing tubes 436 . The impingement fluid enters the collection container 440 . The buffer solution may be pumped from container 420 into collection container 440 . The mixture collected in collection container 440 may persist for a period of time during the incubation. The quench process buffer solution may optionally be maintained in a separate container 450 . The quenched liposome composition 460 exits the collection vessel 440 and enters the filtration process.

In certain embodiments, the buffer solution in container 450 may optionally be used to dilute the impingement fluid and may be contacted with the impingement fluid prior to the collection vessel or prior to any mixing tubes.

As discussed above, to form liposome compositions, certain processes of the present invention provide impingement fluids that undergo mixing in transfer tubes and optionally turbulent mixing tubes, collection in vessels or containers, Incubation process and quenching. The quenched material is exported for filtration and finishing.

The impingement fluid typically results from contacting a composition of active agent with a composition containing liposome-forming molecules. The impingement fluid may only contact the composition to form a single fluid. The impingement fluid typically may not provide complete interdispersion or intermixing of the compositions. In an optional step, the impinging fluid can be subjected to turbulent mixing conditions.

The pH of the impingement fluid can be controlled within a range of about 3 to about 9. In some embodiments, the impingement fluid may have a pH of about 5 to about 8, or about 6 to about 7, or about 7.4. In certain variations, the impingement fluid has a pH of about 3 to about 6. The pH of the impingement fluid can be adjusted during its transfer through the transfer tube or in the mixing tube or in the collection vessel. In certain embodiments, the impingement fluid has an initial pH of about 5 to about 8, alternatively about 6 to about 7.4, or the pH is adjusted to a range of about 3 to about 6 after initial impingement. In certain embodiments, the impingement fluid has a pH of about 7.4 at all times.

The composition used to form the impact fluid may optionally be filtered prior to impact. Compositions of the present invention containing one or more active agents can be filtered by, for example, flow filtration techniques to remove undesired particles or phases having a size greater than about 200 nanometers (nm) or greater than about 300 nm or greater than about 500 nm. Compositions containing various liposome-forming molecules can be filtered by, for example, flow filtration techniques to remove undesired particles or phases having a size greater than about 200 nm or greater than about 300 nm or greater than about 500 nm.

The temperature of the impingement fluid can be controlled in the range of about 15°C to about 37°C.

Aspects of the invention further provide that the impingement fluid composition can be controlled using the fluidity of the impinging composition. Typically, each composition will flow through a tube of selected diameter, so the relative volumetric flow rates of the combined fluids in the impinging fluid provide a means of describing the concentrations of the various components in the impinging fluid.

For example, when an impinging fluid is formed by impinging two separate fluids flowing through a pipe of the same diameter, the mobility of the independent fluids will determine the concentration of the component in the impinging fluid (compared to the concentration of the component in the initial fluid) . Thus, flow rates can be used to create the desired composition in the impingement fluid used to prepare liposome compositions with specific properties.

In certain embodiments, the flow rate of the fluid can be used to control the concentration of active agent relative to liposome-forming molecules, and other parameters including solvent or salt concentration and mixing and shearing forces. Embodiments of the invention include methods for preparing liposome formulations in which active agent encapsulation and liposome particle size are advantageously enhanced by adjusting the flow rate of the processing equipment.

In certain aspects, the methods of the invention may employ tubing of the same diameter used to impinge the fluid of the active agent composition and the fluid of the lipophilic molecule-containing composition. In certain variations, the flow rates of the compositions may be the same or different. In particular embodiments, the flow rate of the active agent-containing composition may not be equal to the flow rate of the lipophilic molecule-containing composition. For example, in certain embodiments, the flow rate of a composition containing an active agent can be twice that of a composition containing a lipophilic molecule. In other variations, the flow rate of the composition containing the active agent may be twice or more that of the composition containing the lipophilic molecule, or in some embodiments, the flow rate of the composition containing the active agent may be Three times or more the flow rate of the composition containing the lipophilic molecule, or five times or more the flow rate of the composition containing the active agent.

In general, the tubing of the device can be of any diameter. In certain embodiments, the methods of the invention may employ tubing of different diameters for impinging the fluid of the active agent composition with the fluid of the composition comprising liposome-forming molecules. In some variations, the diameters of the tubes may be the same or different. For example, in certain embodiments, the diameter of the tube containing the solution of the active agent may be three-quarters the diameter of the tube containing the solution of the lipophilic molecule. In other variations, the diameter of the tube containing the solution of the active agent may be one-half the diameter of the tube containing the solution of the lipophilic molecule. In other variations, the diameter of the tube containing the solution of the active agent may be greater than the diameter of the tube containing the solution of the lipophilic molecule.

In some embodiments, the impinging fluid may be further mixed using some means for flow-through mixing. Flow-through mixing means include mixers having one or more channels, capillaries, or pathways arranged to redirect flow, where the channels, capillaries, or pathways can branch and reconnect one or more times to provide turbulent mixing. Means of flow-through mixing may optionally include mechanical agitators, shakers or stir bars, blades, paddles, disks or blades.

The Reynolds number for turbulent mixing can be greater than 2000, or greater than 2400.

The residence time of the mixing fluid in a turbulent mixer can be controlled by adjusting the flow rate of the impinging fluid. In some variations, the flow rate and residence time of the impinging fluid in the turbulent mixer can be used to control the sizing of liposomal particles.

An example of a turbulent mixing tube for flow-through mixing is a Cole-Parmer Inline Static Mixer K-04669-52, 316 stainless steel tube mixer; 3/16" tube OD, 21 elements.

Vessels, tubes and other flow elements of the apparatus used in the methods of the invention may be fabricated from any material that is inert to the reactants and solvents used and suitable for the reaction conditions such as temperature and pH. Examples of such materials include polymers, metals, stainless steel, glass and ceramics. The vessels, tubes and other flow elements may also be coated with inert substances.

Typically, the vessels, tubes, and other fluid elements are in fluid communication with one or more controllable pumps that can control the flow rates of the solutions and mixtures in the various steps. The device may include various valves, such as a sense valve for controlling flow. The vessels, tubes and other flow elements may be connected with various fasteners which may include ferrules or O-rings. The device may have temperature sensors at various points in the flow path.

The method and apparatus of the present invention can be used as a batch or continuous process.

Collection Vessels, Incubation Processes and Quenching Processes

Referring to FIG. 4 , in some embodiments, the impingement fluid enters a collection container 440 . In collection container 440, the collected impingement fluid enters the incubation process.

The incubation process may include one or more steps of mixing the collected material, one or more steps of diluting with a dilution buffer, one or more steps of adjusting the pH in the collection vessel, and separating the collected material One or more steps of incubation time maintained at a particular temperature.

The collected material can be mixed in the collection vessel using, for example, a mechanical stirrer, shaker or stirring rod, blade, paddle, disk or vane.

In some variations, an impingement fluid can be formed by contacting a composition of active agents with a composition comprising a liposome-forming compound and adding a buffer, solvent, or diluent to the impingement fluid. Addition of buffers, solvents or diluents can be done prior to mixing or collecting the impingement fluid, or can be done in a collection vessel as part of the incubation process. The addition of buffers, solvents and diluents reduces the concentration of active agent, liposome-forming molecules and other components such as another solvent in the collection vessel.

In some embodiments, the concentration of the organic solvent can be diluted to about 50% (v/ v) or less, or about 40% (v/v) or less, or about 35% (v/v) or less, or about 33% (v/v) or less, or about 30% ( v/v) or less, or about 25% (v/v) or less, or about 22% (v/v) or less, or about 20% (v/v) or less.

The pH of the collected mixture or composition in the collection vessel can be controlled within the range of about 3 to about 9. In some embodiments, the pH of the collected impact fluid is adjusted to be in the range of about 5 to about 8, or about 6 to about 7.4. In certain embodiments, the collected mixture has a pH of about 5 to about 8, and the pH is adjusted to be in the range of about 3 to about 6 after the initial impact. In some variations, the pH of the collected impact fluid is maintained at about 7.4. The liposome compositions of the invention can also be formed in a process wherein the pH of each step is about 7.4.

In some aspects, the mixture or composition collected in the collection container undergoes an incubation hold period. The length of the holding period of incubation in the collection vessel can be from minutes to hours, or from about 15 minutes to about 8 hours, or from about 0.5 hours to about 8 hours, or from about 0.5 hours to about 4 hours, or from about 1 hour to about 4 hours. hour, or from about 1 hour to about 2 hours.

In variations of the method, turbulent mixing occurs after dilution of the impinging fluid with a buffer, solvent, or diluent, and the incubation period can be from about 0.5 hour to about 8 hours, or from about 1 hour to about 4 hours. hour, or from about 1 hour to about 2 hours.

In certain variations, turbulent mixing occurs prior to diluting the impinging fluid with a buffer, solvent, or diluent, and the incubation period can range from minutes to hours, or from about 15 minutes to about 8 hours, or from about 0.5 hour to about 8 hours, alternatively about 0.5 hour to about 4 hours, alternatively about 1 hour to about 4 hours, alternatively about 1 hour to about 2 hours.

The length of the incubation period of the incubation process may generally depend on other process parameters such as the flow rate of the impingement stream as well as temperature and pH.

During the holding period, the temperature of the collected composition in the collection vessel can be controlled from about 15°C to about 37°C, or from about 22°C to about 35°C.

In some aspects, the incubation process can be terminated by quenching the incubation by the rapid addition of buffer, solvent, or diluent. The quenching step may reduce the concentration of the organic solvent to about 20% (v/v) or less, or about 15% (v/v) or less, or about 10% (v/v) or less, Or about 5% (v/v) or less.

In some embodiments, the quenching process can further result in a stabilized liposome composition comprising liposomes encapsulating the active agent.

filter and organize

As discussed above, in some embodiments, the impingement fluid undergoes a mixing, collection, incubation process, and quenching process. The quenched material can be a stabilized liposome composition, which can be exported for filtration and finishing.

Figure 5 shows a process diagram for preparing certain embodiments of liposome compositions of the invention by filtration and conditioning. Referring to FIG. 5 , a stabilized liposome composition, such as a quenched liposome composition 460 , is loaded into a container 500 . The diafiltration buffer solution is maintained in a separate container 520 . Peristaltic pump 502 provides circulation of the stabilized liposome composition from container 500 through tube 504 containing hollow fiber membranes. Tangential flow filtration occurs through this cycle to concentrate the stabilized liposome composition, and filtrate 530 is removed from tube 504 containing the hollow fiber membranes. Adding displacement buffer from container 520 to the cycle may allow for diafiltration at fixed or variable volumes. The stabilized liposome composition can also be diluted by the addition of buffering agents to obtain a final concentration of active agent in the formulation. Optionally, dialysis of the stabilized liposome composition can be performed using peristaltic pump 506 to drive dialysis buffer from container 540 . The stabilized liposome composition at a specific concentration is output 550 to a finishing step.

Typically, filtrate 530 may contain organic solvent and unencapsulated active agent. Thus, removal of the filtrate can remove and reduce the concentration of organic solvent in the stabilized liposome composition and remove unencapsulated active agent.

Typically, the concentration of active agent in the quenched incubation may be below the range desired for the preparation of the pharmaceutical composition. In some embodiments, the concentration of the non-aqueous solvent in the quenched incubation may be too high for the preparation of a pharmaceutical composition. These concentrations can be adjusted by tangential flow filtration and diafiltration, as discussed above.

In some embodiments, the quenched incubation is recycled to a hollow fiber tangential flow filtration device or a cartridge or cassette tangential flow filtration device. In the absence of added buffer or solvent circulation, tangential flow filtration retains the liposome composition in a reduced volume of buffer or solvent, thereby increasing its concentration.

Similar equipment can be used in diafiltration mode to remove non-aqueous solvent and replace it with diafiltration buffer. In diafiltration mode, the volume of the circulating retentate is kept substantially constant by adding diafiltration buffer. Therefore, the concentration of the organic solvent is reduced and removed upon entering permeation.

The concentration of active agent can be adjusted to obtain the desired final concentration by adding buffer to the retentate of the diafiltration step. The retentate with adjusted concentration can then be provided to a sterilization unit where the retentate product solution is sterilized using in-line filtration. The sterilized product can be used in an aseptic bottling process and the product bottle is stored at low temperature. Flash freezing, lyophilization, and lyophilization, among other means, can be used to prepare and preserve the product.

In certain embodiments, to achieve the desired concentration of active agent, one may first pass tangential flow filtration, then diafiltration to remove organic solvents, then pass additional tangential flow filtration and finally wash with buffer, solvent or diluent. Dilute to concentrate the quenched incubation.

Examples of methods and materials for filtration are given in Mark C. Porter, Handbook of Industrial Membrane Technology (Noyes 1990), pp. 186-87. Some aspects of filtration are given in Munir Cheryan, Ultrafiltration and Microfiltration Handbook (1998).

Packing of Active Agents

The degree of encapsulation of active agents by liposome particles is generally influenced by several process parameters.

In some embodiments, the degree of encapsulation of the active agent by liposome particles after the incubation process is 50% or higher, or 60% or higher, or 70% or higher, or 80% or higher, or 90% or higher. % or higher, or 95% or higher, or 96% or higher, or 97% or higher, or 98% or higher, or 99% or higher, or substantially 100%.

The active agent liposome compositions of the invention generally comprise liposomal particles of uniform size. The liposome particle size diameter may be about 300nm or lower, or about 250nm or lower, or about 200nm or lower, or about 180nm or lower, or about 160nm or lower, or about 150nm or lower, or about 140 nm or less, or about 130 nm or less, or about 120 nm or less, or about 110 nm or less, or about 100 nm or less, or about 90 nm or less, or about 80 nm or less, or About 70nm or less.

The liposome particle size may range from about 50 nm to about 500 nm, or from about 60 nm to about 400 nm, or from about 70 nm to about 300 nm, or from about 70 nm to about 200 nm, or from about 70 nm to about 160 nm, or from about 80 nm to about 160 nm .

In some variations, the stabilized liposome composition may comprise less than about 10% active agent outside of the liposome particle and unencapsulated, or less than about 8% active agent unencapsulated, or Less than about 5% unencapsulated active agent, or less than about 4% unencapsulated active agent, or less than about 3% unencapsulated active agent, or less than about 2% unencapsulated active agent, or less than about 1% unencapsulated active agent.

The level of entrapment of the active agent in the stabilized liposome composition can be from about 70% to about 99%, alternatively from about 80% to about 99%, alternatively from about 90% to about 99%, alternatively from about 95% to about 99%. The level of entrapment of the active agent in the stabilized liposome composition can be substantially 100%.

Efficient delivery of gene silencing therapeutics

The present invention relates generally to novel compounds and compositions for the delivery of biologically active and pharmaceutical agents and methods and uses thereof. The compounds and compositions of the invention are useful for delivering therapeutic agents to selected cells, tissues, organs or subjects. More specifically, the invention relates to the delivery of therapeutic agents, including nucleic acid agents, and methods for making and using peptide-containing substances for the delivery of biologically active and pharmaceutical agents.

The present invention provides various compounds, compositions, methods and uses for effective systemic and local delivery of drugs and biologically active molecules. Efficient delivery can be achieved by high loading of active agents into liposomes using a variety of carrier molecules including peptides. The compounds and compositions of the present invention allow for efficient delivery of active agents.

One calculation of delivery efficiency is the delivery efficiency ratio. As used herein, the delivery efficiency ratio is the ratio of the total mass of carrier molecules to the mass of active agent. The lower the delivery efficiency ratio, the lower the quality of the carrier material compared to the active agent, and the lower the potential for undesired toxicity and side effects. As used herein, a lower delivery efficiency ratio is more beneficial and more desirable.

The present invention relates generally to the field of vectors and formulations for nucleic acid delivery. Vectors for nucleic acids include compounds and compositions formed with peptide components including crosslinkable and cleavable peptide structures. More specifically, the invention provides crosslinkable peptide structures and cleavable peptide structures that bind to nucleic acids to form complexes or condensed compositions.

In some embodiments, the invention provides complexes formed from peptides and nucleic acids. These complexes comprise a core structure with a complex of peptide and nucleic acid and a core structure with multiple peptide and nucleic acid layers. Peptides suitable for forming complexes of the invention with nucleic acids include any cationic peptide.

In some aspects, complexes, condensates or nanoparticles of peptides and nucleic acids can be loaded into liposome lipids. The liposome formulations of the present invention can provide a stable delivery system for bioactive agents and pharmaceutical agents, especially nucleic acid agents.

In some aspects, the compositions and formulations of the invention can provide biological activity with reduced toxicity.

The invention also provides methods for using the vectors, peptides, nucleic acid constructs or complexes with peptides and formulations of the invention (optionally in combination with cell targeting components and other pharmaceutical formulation components) in altering gene expression or activity. method.

The present invention provides various carrier compositions for delivering biologically active agents to cells. More specifically, the present invention provides a variety of carrier structures in which nucleic acid reagents are condensed into small particles, which are nanoscale in size. The carrier particles can have increased stability in delivery and can effectively deliver the active agent. Formulations of carrier particles loaded into liposomes can provide improved stability and delivery efficiency.

The novel compounds and compositions of the present invention can achieve a series of favorable delivery efficiency ratios. In some embodiments, compositions of the invention provide a delivery efficiency ratio of less than 15 or less than 10 for RNAi-inducing agents.

The compositions and methods of the invention are useful for the delivery of therapeutic, prophylactic, and diagnostic agents, such as nucleic acids, polynucleotides, peptides, proteins, and small molecule compounds and drugs. These compositions can include nanoparticles of various diameters.

The present invention provides novel compounds, compositions and formulations for the intracellular and in vivo delivery of active agents that generally maintain cytoprotective effects and are relatively low toxic for ultimate use as therapeutic agents. The compounds and compositions of the invention are useful for the delivery of active agents into selected cells, tissues, organs or compartments for the purpose of altering a disease state or phenotype.

In some aspects, the invention provides compounds, compositions and methods for delivering RNA constructs to cells to produce RNA interference responses, antisense effects, or modulation or modulation of genomic expression.

In some variations, the present invention provides compounds, compositions and methods for delivering DNA constructs or DNA-containing substances to cells.

As used herein, the term "peptide nucleic acid complex" refers to a peptide bound or complexed to a nucleic acid.

Efficient delivery of antisense reagents that induce RNAi

In some aspects, the compositions and methods of the invention provide efficient delivery of active agents by providing carrier particles with a high concentration or density of active agent molecules. Carrier particles can be loaded into liposomes to provide high concentrations or densities of active agent molecules in pharmaceutical formulations.

In certain aspects, the compositions and methods of the invention provide formulations of RNAi-inducing or antisense agents with various delivery efficiency ratios. The delivery efficiency ratio of the formulation for RNAi-inducing agent or antisense agent of the present invention may advantageously be less than 15, or less than 12, or less than 10, or less than 9, or less than 8, or less than 5.

In certain embodiments, efficient delivery can be achieved using carrier particles composed of nucleic acid agents condensed with cationic peptides. For example, based on the charge of a cationic peptide combined with a nucleic acid agent (eg, RNAi-inducing agent or antisense agent), the carrier particle can include a structure in which up to 6 or more peptide binding regions can bind to an active RNA agent.

In some variations, in formulations containing carrier particles consisting of nucleic acid agents (e.g., RNAi-inducing agents or antisense agents) loaded into liposomes, each particle of liposomes may have greater than 500 or greater than 1,000 Or greater than 5,000 or greater than 6,000 or greater than 7,000 or greater than 8,000 or greater than 9,000 or greater than 10,000 or more copies of RNAi-inducing agent or antisense agent molecules.

For example, in some aspects, for a N:P of 2, a density of 1 g/cc, a particle volume of 1.26 x ^{106 nm3} , and a peptide of MW 3781.2 (7 net positive charges) and double-stranded RNA of MW 13,500 (40 net negative charge), the particle mass is 1.26×10 ^-9 μg, and each double-stranded RNA of the particle has 11.4 peptides. In this example, the delivery efficiency ratio, the ratio of peptide mass to RNA mass, was 3.2. The mass fraction of particles represented by RNA was 0.24, the number of double-stranded RNA molecules in the particles was 13,369, and the number of peptide molecules per particle was 1.53×10 ⁵ . In other words, the formulation containing these carrier particles without additional carrier molecules had a delivery efficiency ratio of 3.2 and a particle-based RNAi agent mass fraction loading of 24%.

Liposome formulation

In some aspects, carrier particles of the invention can be loaded or encapsulated in liposomal formulations. For example, in some embodiments, carrier particles can be delivered encapsulated in a liposomal formulation (eg, as disclosed in US Patent Application Serial No. 12/114,284).

In certain embodiments, pharmaceutical formulations of the carrier particles of the invention delivered encapsulated in liposome formulations can reduce the loading of double-stranded RNA compared to RNA liposome formulations without the peptide carrier particle compositions of the invention. volume increased by 20 times.

For example, in some embodiments, compared to RNA liposome formulations without the peptide carrier particle composition of the present invention, pharmaceutical formulations of the carrier particles of the present invention delivered encapsulated in liposome formulations can reduce the amount of carrier substance 45% lower.

In some embodiments, pharmaceutical formulations of carrier particles for RNA agents include peptide-containing delivery systems that use peptides to deliver nucleic acids. This system can increase the loading of RNA agents that can be incorporated into liposome formulations. The efficiency of delivery as well as the tissue distribution pattern of the delivery system can be enhanced using nanoparticles comprising peptides. In some embodiments, the delivery system can demonstrate up to a 20-fold increase in RNA loading per liposome particle while reducing the total amount of carrier excipient by about 45%. In some variations, such as calculated by in vivo knockdown of ApoB, the system can achieve a 30% reduction in RNA agents compared to liposomal formulations without peptide, while neutralizing mouse liver 85% knockdown was maintained in the jejunum. Thus, the pharmaceutical formulation of the carrier particles of the present invention can significantly increase the delivery efficiency of RNA agents such as siRNA, mdRNA or antisense agents.

carrier nanoparticles

In some embodiments, the carrier particles of the present invention can be prepared as described in US Patent Application 61/106,062, filed October 16, 2008.

The carrier particles of the present invention are generally of uniform size. The carrier particle size diameter can be about 300nm or lower, or about 250nm or lower, or about 200nm or lower, or about 180nm or lower, or about 160nm or lower, or about 150nm or lower, or about 140nm or less, or about 130nm or less, or about 120nm or less, or about 110nm or less, or about 100nm or less, or about 90nm or less, or about 80nm or less, or about 70nm or lower.

The particle size of the active agent carrier particles of the present invention may range, for example, from about 50 nm to about 500 nm, or from about 60 nm to about 400 nm, or from about 70 nm to about 300 nm, or from about 70 nm to about 200 nm, or from about 70 nm to about 160 nm, or About 80nm to about 160nm.

In some embodiments, the active agent carrier particles of the present invention can be negatively charged. For example, a carrier particle composed of an RNAi agent and a cationic peptide can be condensed such that the particle retains a negative charge.

In some embodiments, the active agent carrier particles of the present invention can be positively charged. For example, a carrier particle consisting of an RNAi agent and a cationic peptide can be condensed such that the particle acquires a positive charge.

Carrier and peptide binding domain

In some aspects, the carrier compounds and compositions of the invention can be formed with peptide components that condense with biologically active nucleic acid components by binding to the nucleic acids to form nano-sized particles.

In some embodiments, peptides suitable for the vector compositions of the present invention are described in US Patent Application 61/116,258, filed November 19, 2008.

When a peptide having one or more binding domains is bound to a nucleic acid, a vector can be formed.

In certain variations, more than one cation-binding region of a peptide can bind to the same or different nucleic acid molecules.

The crosslinkable and cleavable peptide constructs of the invention may advantageously have a plurality of cationic residues distributed along the peptide chain within one or more binding regions. Variations in the number and distribution of cationic residues can be used to alter the strength of binding of the peptide to the active agent.

Peptides of the invention include cationic peptides having a binding region sufficiently positively charged to bind nucleic acids and one or more linker groups. The binding region of the peptide of the present invention may have a sufficient positive charge to bind nucleic acid. Linker groups can be linked to each other to cross-link two or more peptides within a single molecule.

A peptide of the invention capable of condensing with an active nucleic acid reagent to form a carrier particle may have a sufficient positive charge to bind nucleic acid and a linker group sufficient to form a construct comprising self-crosslinking nucleic acid binding.

The present invention provides peptides having sufficient positively charged residues to bind nucleic acids and capable of forming self-crosslinking peptides.

In some embodiments, the bioactive agent is a nucleic acid agent capable of binding a cationic peptide. A nucleic acid agent can bind 1, 2, 3, 4, 5 or 6 peptides or more to form a complex. Condensate particles can be formed by polymerization and association of nucleic acid-peptide complexes.

In some embodiments, a nucleic acid reagent can bind a moiety with more than one peptide such that the peptides are linked to more than one nucleic acid reagent.

A carrier structure or construct can be formed by mixing a crosslinkable or cleavable peptide of the invention and a bioactive agent bound to the peptide. Binding of the peptide to the reagent can occur at the same time as the crosslinking of the peptide occurs, or before or after the peptide is crosslinked.

In some aspects, the carrier is a cross-linked peptide construct, which may be a condensate of a peptide and a nucleic acid. The condensates can form nano-sized carrier particles that can incorporate bioactive agents such as nucleic acids.

cross-linkable peptide

In some embodiments, a crosslinkable peptide of the invention may comprise a crosslinkable terminal residue or group.

For example, a cross-linkable peptide can have a single terminal cysteine residue that can be cross-linked by forming an inter-peptide disulfide bond (forming a dimer of the peptide).

In some variations, the peptide can comprise one or more sulfhydryl groups that can be cross-linked to form a polypeptide construct, wherein the polypeptide construct is conjugated to a bioactive agent and can be a bioactive agent. carrier.

In some embodiments, a crosslinkable group can form a cleavable crosslink that can be cleaved at low pH or can be cleaved by the action of a protein or an enzyme. Examples of cleavable crosslinks include chemically cleavable acid labile crosslinks and enzymatically cleavable crosslinks.

Examples of crosslinkable groups include organic groups of up to 1000 atoms, bifunctional linkers, bifunctional crosslinkers, and heterobifunctional linkers. The crosslinkable group may be a substituent of a peptide residue or may be attached to a terminus of the peptide.

In certain embodiments, crosslinkable peptide structures include peptides with crosslinkable groups at each terminus. In some variations, crosslinkable peptide structures include dimers, trimers, and multimers of peptides with crosslinkable groups at each terminus.

cleavable peptide

In some aspects, the invention provides cleavable peptides containing internal cleavable linker groups located between portions of the peptide sequence.

In some embodiments, a cleavable peptide can have two cation-binding regions linked together by a cleavable group. The cleavable group can be cleaved to separate the binding regions of the peptide from each other.

The cation binding region can bind biologically active agents such as nucleic acids.

In some variations, cleavage of the peptide's linker group that separates the binding region may allow for more rapid dissociation of the peptide from the bioactive agent compared to a non-cleavable peptide.

Intracellularly cleavable linkers can be cleaved by chemical reduction or by the action of various proteins or enzymes in the intracellular environment.

Condensate granules and releasable forms

The compounds and compositions of the invention include condensate particles or carriers consisting of one or more peptide components and one or more active agents.

In general, the condensate particles formed from the peptide and the active agent can be anionic, neutral or cationic. For in vivo delivery of carrier particles, neutral or cationic forms may be preferred. The condensate particles may be referred to as core particles.

In some embodiments, a first portion of a crosslinkable peptide and an active agent can be used to form condensate particles. One or more additional layers of the same or different crosslinkable peptides may be added to the particle.

In some variations, the first portion of the cleavable peptide and the active agent can be used to form condensate particles. One or more additional layers of the same or different cleavable peptides may be added to the particle.

In certain embodiments, a first portion of a cleavable peptide and an active agent can be used to form a condensate particle. One or more additional layers of crosslinkable peptides may be added to the particle.

In some variations, the first portion of the crosslinkable peptide and the active agent can be used to form condensate particles. One or more additional cleavable peptide layers may be added to the particle.

In some embodiments, an anionic condensate particle can be formed with a first portion of a crosslinkable or cleavable peptide and an active nucleic acid agent. One or more additional layers of cationic crosslinkable or cleavable peptides may be added to the anionic particles to form neutral or cationic carrier particles.

In certain variations, an anionic condensate particle can be formed with a first portion of a crosslinkable or cleavable peptide and an active nucleic acid agent. One or more additional layers of cationic crosslinkable or cleavable peptides may be added to the anionic particles to form neutral or cationic carrier particles. One or more additional layers of anionic endosomolytic compounds may be added to the neutral or cationic carrier particles to form neutral or cationic carrier particles.

In some aspects, the active agent can be one or more pharmaceutical compounds, one or more antisense agents, one or more RNAi-inducing agents, or one or more DNA-containing agents.

In some embodiments, the compositions or formulations of the invention can be prepared by loading condensate particles or layered carrier particles into cationic liposomes.

In some embodiments, the compositions and methods of the invention can provide for the delivery of a therapeutic agent in a releasable form or composition. Releasable forms or compositions include molecules that bind and release the active agent, molecules that bind the active agent and unload moieties that facilitate release of the agent, molecules that bind the active agent and are then within the biological compartment to facilitate release of the agent Mode modulating molecules, and compositions comprising molecules combined with active agents mixed with release mediating compounds.

As used herein, releasable forms include forms comprising crosslinkable or cleavable peptides of the invention, or forms comprising endolysosomal compounds or substances.

Adducts or carrier particles may comprise cleavable peptide structures or matrices. Certain events, such as entry of the vector into a biological environment or compartment containing a compound capable of cleaving crosslinks of the peptide, may trigger cleavage of the peptide structure. Cleavage of the peptide linker group can occur intracellularly in the cytosol or within various cellular or extracellular compartments.

Cleavage of the disulfide peptide linker group can occur chemically, for example by reduction of the disulfide with tris(2-carboxyethyl)phosphine hydrochloride (TCEP), dithiothreitol (DTT), or mercaptoethanol And proceed.

In certain embodiments, peptide disulfide bonds can be cleaved using a disulfide reductase enzyme.

Once inside the cell, the disulfide cross-links can be reduced, thereby releasing the active agent for efficient delivery. The environment of the endosome is believed to be reducing and mediates the reduction of the disulfide and the release of the active agent.

Release can occur intracellularly by cleavage or cleavage of peptide cross-links, as well as by dissociation of the bioactive agent from the peptide.

The peptides and peptide constructs of the invention may advantageously comprise 1, 2 or more binding regions with 1 or more positively charged amino acid residues. The binding domains may be linked in a chain in which one positively charged binding domain is cleavably linked to the next binding domain by a cleavable cross-link.

The cationic region can serve as a binding region for an active agent such as a nucleic acid agent, and several cationic regions can bind to the same active agent to collectively link the peptide to the active agent.

In certain embodiments, releasable forms of the invention comprise particles of condensates of peptides and nucleic acids, wherein the peptide component includes crosslinks that can be cleaved to effect release of the nucleic acids. Cleavage of the linker group of the peptide may be triggered by a change in the peptide environment, for example during trafficking from the extracellular to the intracellular domain, or during endocytosis or uptake and delivery of endosomes by the cell The change.

Examples of cleavable linkers for peptides include acid cleavable groups such as hydrazones that are cleavable during endocytosis or by intracellular interactions with lysosomes.

In some embodiments, the release of the active agent can occur from an acid labile linker.

Examples of acid labile linkers include those containing orthoester groups, hydrazones, cis-acetylmethyl, acetals, ketals, silyl ethers, silazanes, imines, citric anhydride, horses Linkers for toric anhydride, crown ether, azacrown ether, thiacrown ether, dithiobenzyl group, cis-aconitic acid, cis-carboxyalkatriene, methacrylic acid, and mixtures thereof.

Examples of acid labile groups and linkers are described in US Pat.

Examples of cleavable linkers for peptides include cathepsin-cleavable linkers, such as Val-Cit, which can be cleaved by intracellular cathepsins. Examples of substrate sequences for cathepsins B, D and L are shown in Tables 1, 2 and 3, respectively. Cleavable linkers include dipeptide, tripeptide and tetrapeptide subunits (P2-P2') of cathepsin B, D and L substrates.

Table 1: Cathepsin B Substrates

Table 2: Cathepsin D Substrates

Table 3: Cathepsin L Substrates

In some variations, the releasable forms of the invention include particles of condensates of peptides and nucleic acids and endolysosomal compounds. In these variations, the endolysomal compound may aid in the release of the core particle and active agent from the endosome into the cell, while the peptide component may include a compound that can be cleaved to effect the release and dissolution of the nucleic acid from the core condensate particle within the cell. isolated cross-linking.

Examples of endolysosomal compounds include chloroquine, 4-aminoquinoline, aminoquinoline, amodiaquine, cell penetrating peptide, transporter (Transportan), penetratin, hemagglutinin fusion peptide from influenza virus (see, For example, Han et al., Nat. Struct. Biol. Vol. 8, 715-720, 2001) and the influenza virus-based peptide diINF7.

In certain embodiments, carrier particles or constructs can be formulated with targeting agents for cellular or subcellular delivery. In some variations, carrier particles can be combined with synthetic polymers such as polyethylene glycol (PEG) to reduce non-specific effects or interactions with blood components. Suitable synthetic polymers include polyethylene glycol chains (PEG) or PEG copolymers, such as PEG-polyurethane or PEG-polypropylene. See, eg, J. Milton Harris, Poly(ethylene glycol) chemistry: biotechnical and biomedical applications (1992).

Instructions

The invention includes a method for delivering a therapeutic nucleic acid to a cell comprising preparing a composition comprising carrier particles comprising a nucleic acid agent and treating the cell with the composition.

The invention includes a method for inhibiting gene expression in a cell comprising preparing a composition comprising carrier particles comprising a nucleic acid agent and treating the cell with the composition.

The invention includes a method for inhibiting gene expression in a mammal comprising preparing a composition comprising carrier particles comprising a nucleic acid agent and administering the composition to the mammal.

The present invention includes a method for treating a human disease selected from inflammatory diseases including rheumatoid arthritis, metabolic diseases including hypercholesterolemia, liver disease, encephalitis, bone fractures, heart disease, including hepatitis and influenza viral diseases and cancer; the method comprising preparing a liposome composition and administering the composition to a human.

active agent

In some aspects, the invention provides methods for preparing compositions suitable for the delivery of therapeutic agents. The methods of the invention can provide nucleic acid reagent compositions such as condensed RNA nanoparticles, double- or triple-stranded RNA structures, RNA peptide conjugates, Dicer substrate RNA, dsRNA, siRNA, microRNA, hairpin RNA, and Other active regulatory RNA forms, antisense therapeutic forms including antisense RNA and antisense DNA, and DNA and DNA-containing forms.

The active agents of the invention can be single-stranded or double-stranded nucleic acids. The active agent of the invention may be an antigenic or immunogenic protein or polypeptide.

The active agent of the invention may be a peptide condensate of the active agent. For example, the active agent may consist of nanoparticles formed by condensation of the active agent with a peptide or other biomolecule, or of a condensate or complex of the active agent with a peptide, biomolecule, or polymeric molecule. Nanoparticles or condensates can be crosslinked. Nanoparticles or condensates can be loaded into liposome compositions as loads.

Active agents of the invention may be antisense or sense DNA or RNA oligonucleotides, or modified DNA or RNA oligonucleotides, which bind to a target nucleic acid sequence through a variety of interactions to block transcription or translation of the target sequence . An antisense or sense agent may form a triple helix with a nucleotide duplex, or may be a ribozyme, or may encode a transcriptional or translational regulatory sequence including a promoter sequence or enhancer sequence. Antisense or sense oligonucleotides can be used to block protein expression and can have modified nucleobase or sugar groups, or other groups, or can be conjugated to biomolecules, peptides or proteins, with to enhance stability or activity. Antisense or sense oligonucleotides can be delivered to cells containing their target nucleic acids by the compositions and methods described herein. As described herein, antisense or sense oligonucleotides can be delivered to cells containing their target nucleic acids using oligonucleotide-carrier complexes or liposome formulations.

Cross-linkable and cleavable peptides

The cross-linkable peptides of the present invention include cross-linkable peptides having the structure shown in formula I:

A-B Formula I

Wherein, A is a peptide of 2 to about 16 amino acid residues, which may contain a cation binding region, and B is a cross-linkable group, wherein A contains one or more positively charged residues at pH7.

Examples of B include cysteine.

Other examples of B include organic groups with up to 1000 atoms, bifunctional linkers, bifunctional crosslinkers and heterobifunctional linkers, carbamates and esters.

Examples of A include cationic peptides.

Examples of A include cationic peptides having the structure shown in Formula II:

(Xaa ¹ ) _m -(Xaa ² ) _n -(Xaa ³ ) _o -(Xaa ⁴ ) _p Formula II

Wherein Xaa is an amino acid residue, each of Xaa ¹ , Xaa ² , Xaa ³ and Xaa ⁴ is the same or different amino acid residue selected independently, each of m, n, o and p is 0 to 4, as long as m, n The sum of , o and p is 2 or higher, wherein one or more of Xaa ¹ , wXaa ² , Xaa ³ and Xaa ⁴ are positively charged residues at pH7.

Cationic peptides can be prepared in which residues such as A have basic side chains. Examples of amino acids with basic side chains include arginine (Arg), homoarginine (homoArg) (side chain-( _CH2 ) _4NH (C=NH) _NH2 ), nor-arginine (nor Arg) (side chain-(CH ₂ ) ₂ NH(C=NH)NH ₂ ), n-nor-arginine (n-n-Arg) (side chain-(CH ₂ )NH(C=NH)NH ₂ ), Ornithine, Lysine, Homolysine, Histidine, 1-Methylhistidine, Pyridylalanine (Pal), Asparagine, N-Ethylasparagine, Glutamine and 4-Aminophenylalanine and its side chain modified derivatives.

As used herein, the term "high", when referring to an amino acid, means that an extra carbon is added to the side chain, and the term "normal", when referring to an amino acid, means that a carbon is subtracted from the side chain. Thus, homolysine represents the side chain -(CH ₂ ) ₅ NH ₂ .

Cationic peptides can also be prepared in which the side chains of the residues contain ionizable genes or substituents.

In some embodiments, the cationic residue shown is NG ^- methylarginine, symmetric or asymmetric ^NG , ^NG -dimethylarginine, ^NG -methyl-homoarginine, Symmetrical or asymmetrical ^NG , NG ^- dimethyl-homoarginine, NG ^- methyl-norarginine, Symmetrical or asymmetrical ^NG , NG ^- dimethyl-norarginine or N ^G -methyl-nor-nor-arginine, symmetrical or asymmetrical N ^G , N ^G -dimethyl-nor-nor-arginine.

In some embodiments, the cationic residue shown is NG ^- ethylarginine, symmetric or asymmetric NG, ^NG - ^{diethylarginine} , NG ^- ethyl-homoarginine, Symmetrical or asymmetrical ^NG , NG- ^diethyl -homoarginine, NG ^- ethyl-norarginine, Symmetrical or asymmetrical ^NG , NG- ^diethyl -norarginine Or NG ^- ethyl-nor-nor-arginine, symmetrical or asymmetrical ^NG , ^NG -diethyl-nor-nor-arginine.

In certain embodiments, the indicated cationic residue is NG ^- alkylarginine, symmetric or asymmetric ^NG , ^NG -dialkylarginine, NG ^- alkyl-homoarginine , symmetric or asymmetric ^NG , NG ^- dialkyl-homoarginine, NG ^- alkyl-norarginine, symmetric or asymmetric ^NG , NG ^- dialkyl-norarginine acid or ^NG -alkyl-n-n-arginine, symmetric or asymmetric ^NG , ^NG -dialkyl-n-n-arginine.

In some embodiments, the cationic residue shown is an amino acid with a guanidine- or amidine-containing side chain. For example, the side chain of an Xaa residue may contain compounds such as guanidino, amidino, dihydroimidazole, 4-guanidino-phenyl, 4-amidino-phenyl, N-amidino-piperidine, N-amidino- A group of piperazine, 4,5-dihydroimidazole, 2-(N-amidino)-pyrrolidinyl or 4-[(2-aminopyrimidinyl)]ethyl.

Examples of cationic residues can have side chains including the following structures and their salt forms:

The cleavable peptides of the present invention include dimer peptides of the structure shown in formula I, such as dimer A-B-B-A, wherein the linker groups B are capable of linking to each other, and wherein the -B-B- link is cleavable.

For example, the dimer A-B-B-A can be A-B-(S-S)-B-A, where (S-S) is a disulfide bond.

Other examples of -B-B- linkages include organic groups with up to 1000 atoms, linkages formed by bifunctional linkers, linkages formed by bifunctional crosslinkers and linkages formed by heterobifunctional linkers, hydrazine linkers, Urethane linkages and ester linkages.

The cross-linkable peptides of the present invention include cross-linkable peptides having the structure shown in formula II:

B-A-B Formula II

wherein A is a peptide of 2 to about 16 amino acid residues and B is a crosslinkable group as defined above, wherein A contains one or more positively charged residues at pH7.

Examples of B include cysteine.

Examples of A include cationic peptides.

The cleavable peptides of the present invention include dimers, trimers or multimers of the structure shown in formula II, such as dimer BABBAB and multimer -(BAB) _n- , wherein the linker group B can interact with each other connection, and where the -BB- connection is cleavable. Some of these cleavable peptides remained crosslinkable due to their retained crosslinkable groups at each terminus.

Examples of cation-binding domains suitable for preparing peptides of the invention are shown in Table 4. The cross-linkable peptides of the present invention may have the binding regions shown in Table 4 with cysteine linked to the N-terminal or C-terminal of the peptides shown in FIG. 4 . The crosslinkable peptides of the invention can form dimers.

Table 4: Binding regions used to prepare peptides

SEQ ID NO: binding region 195 GRKKRRQRRRPPQ 196 KKKRKV 197 KKKRKVKKKRKV 198 GRKKRR 199 RRRPPQ 200 WKKKK 201 RRRPPQH 202 KKRRQH 203 RRR 204 RRRR 205 RRRRR 206 KKK 207 RRRRWW 208 RRRWW 209 RRWW 210 KKWW 211 KKKWW 212 WHHRRKK 213 RRKKHHWW 214 KKRRW 215 KKRRHW 216 KKRRHHW 217 KKRRQ

SEQ ID NO: Binding region 218 KKRRQ 219 GRKKRRQ 220 QGRKKRR 221 RRH 222 RRRH 223 RRRRH 224 RRRRH 225 KKH 226 KKKH 227 HWKKRR 228 HWKKRR 229 PPHRRR 230 PPHRRR 231 GRKKRRVRRRPPQ 232 WWHHKKRRGGRRKKHHWW 233 WWHHKKRR 234 YYHHKKRR 235 RRKKHHYY 236 VQAAIDYING 237 WWRRHH 238 HHRRWW 239 YYRRHH 240 HHRRYY 241 WWRRR 242 RRRWW 243 YYRRR 244 RRRYY 245 WWRRRHH

SEQ ID NO: Binding region 246 HHRRRWW 247 YYRRRHH 248 HHRRRYY 249 WWRRRR 250 RRRRWW 251 YYRRRR 252 RRRRYY 253 WWRRRRHH 254 HHRRRRWW 255 YYRRRRHH 256 HHRRRRYY 257  WWHH-Orn-Orn-RR 258 WWHHHRRR 259 WWHHHRRR 260 WWWHHHHRRR 261 WWWKKRRR 262 KKKWRRW 263 WRRRWRR 264 WWHHKKRR 265 WWCHHKKCRR 266 WWHHHRRR 267 WWHHCKKRR 268 WWHHKKCRR 269 RRWWKKHH 270 WWHHKKKK 271 WWHHRRRR 272 RRRRHH 273 HHKKKK

SEQ ID NO: binding region 274 HHRRRR 275 YYRRRRHH 276 YYKKKKHH

Examples of cleavable peptides of the present invention are shown in Table 5.

Table 5: Cleavable Peptides

SEQ ID NO: peptide 277 GRKKRRV-Cit-RRRPPQ 278 GRKKRRV-Cit-RRKKRG 279 RRRPPQV-Cit-PPRRR 280 RRKKRGV-Cit-GRKKRR 281 QPPRRRV-Cit-RRRPPQ 282 WKKKKV-Cit-KKKKW 283 KKKKWV-Cit-WKKKK 284 HQPPRRRV-Cit-RRRPPQH 285 QPPRRRV-Cit-RRRPPQ 286 HQRRKKV-Cit-KKRRQH 287 RRV-Cit-RR 288 RRRV-Cit-RRR 289 RRRRV-Cit-RRRR 290 RRRRRV-Cit-RRRRR 291 KKV-Cit-KK 292 KKKV-Cit-KKK 293 KKKKV-Cit-KKKK 294 KKKKKV-Cit-KKKKK 295 WWRRRRV-Cit-RRRRWW 296 WWRRRV-Cit-RRRWW 297 WWRRV-Cit-RRWW

SEQ ID NO: peptide 298 WWKKV-Cit-KKWW 299 WWKKKV-Cit-KKKWW 300 WWKKKKV-Cit-KKKKWW 301 KKRRHHWV-Cit-WHHRRKK 302 WWHHKKRRV-Cit-RRKKHHWW 303 WRRKKV-Cit-KKRRW 304 WHRRKKV-Cit-KKRRHW 305 WHHRRKKV-Cit-KKRRHHW 306 QRRKKV-Cit-KKRRQ 307 KKRRQV-Cit-QRRKK 308 RRKKRGV-Cit-GRKKRR 309 GRKKRRV-Cit-RRKKRG 310 QRRKKRGV-Cit-GRKKRRQ 311 QGRKKRRV-Cit-RRKKRGQ 312 HRRV-Cit-RRH 313 HRRRV-Cit-RRRH 314 HRRRRV-Cit-RRRRH 315 HRRRRRV-Cit-RRRRH 316 HKKV-Cit-KKH 317 HKKKV-Cit-KKKH 318 HKKKKV-Cit-KKKKH 319 HKKKKKV-Cit-KKKKKH 320 HWKKRRV-Cit-RRKKWH 321 RRKKWHV-Cit-HWKKRR 322 PPHRRRV-Cit-RRRHPP 323 RRRHPPV-Cit-PPHRRR 324 YYHHKKRRC-Disulfide bond-CRRKKHHYY 325 YYHHKKRRV-Cit-RRKKHHYY

SEQ ID NO: peptide 326 WWRRC-Disulfide Bond-CRRWW 327 WWRRV-Cit-RRWW 328 YYRRC-disulfide bond-CRRYY 329 YYRRV-Cit-RRYY 330 WWRRHHC-Disulfide-CHHRRWW 331 WWRRHHV-Cit-HHRRWW 332 YYRRHHC-disulfide bond-CRRHHYY 333 YYRRHHV-Cit-RRHHYY 334 WWRRRC-Disulfide Bond-CRRRWW 335 WWRRRV-Cit-RRRWW 336 YYRRRC-disulfide bond-CRRRYY 337 YYRRRV-Cit-RRRYY 338 WWRRRHHC-Disulfide-CHHRRRWW 339 WWRRRHHV-Cit-HHRRRWW 340 YYRRRHHC-disulfide bond-CRRRHHYY 341 YYRRRHHV-Cit-RRRHHYY 342 WWRRRRC-Disulfide Bond-CRRRRRWW 343 WWRRRRV-Cit-RRRRWW 344 YYRRRRC-disulfide bond-CRRRRYY 345 YYRRRRV-Cit-RRRRRYY 346 WWRRRRHHC-Disulfide-CHHRRRRWW 347 WWRRRRHHV-Cit-HHRRRRWW 348 YYRRRRHHC-disulfide bond-CRRRRHHYY 349 YYRRRRHHV-Cit-RRRRHHYY 350 WWHHKKRRWV-Cit-WRRKKHHWW 351 WWHH-Orn-Orn-RRV-Cit-RR-Orn-Orn-HHWW 352 WWHHC-Disulfide Bond-CKKRR

Amino acid names and designations as used herein refer to any stereoisomer of the corresponding amino acid.

In Table 5, groups within the peptide sequence may provide cleavage sites. For example, the internal cleavage site can be a disulfide bond or a Val-Cit linkage.

Examples of cleavable linkages include Phe-Lys, Val-Cit, Ala-Leu, Leu-Ala-Leu, and Ala-Leu-Ala-Leu (SEQ ID NO: 376), as described in US Patent Publication 20080166363.

Route of administration

The active agent compositions of the present invention may be used in pharmaceutical compositions. Administration of the liposomal formulations of the invention to a subject can be parenteral, oral, by inhalation, topically, mucosal, rectal or buccal. Parenteral applications include subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrastemal, intrathecal, intralesional, intracranial injection or infusion techniques.

effective amount

An effective amount of an active agent composition of the invention for use in treating a particular disease is generally an amount sufficient to ameliorate or alleviate the symptoms of the disease. An effective amount of an active agent composition of the invention may be an amount sufficient to elicit any biological effect produced by the agent. The composition may be administered as a single dose, or may be administered in repeated doses.

DILA2 amino acid liposome forming compound

The liposome composition of the present invention may include one or more DILA2 amino acid compounds disclosed in US 2008-0317839 A1.

DILA2 amino acid compound is a synthetic organic compound that can form a liposome structure under certain conditions. DILA2 amino acid compounds can be formed by substituting a delivery enhancing or lipophilic tail at the N-terminal or C-terminal or both ends of the amino acid. In some embodiments, the amino acid core may comprise one or more amino acids, or may be a peptide of 2 to 20 amino acid residues.

DILA2 amino acid compounds can be cationic or non-cationic, where non-cation includes neutral or anionic. As used herein, the physiological state or ionicity of a substance refers to an environment with a pH of about 7, unless otherwise indicated.

In some aspects, DILA2 amino acid compounds can provide for delivery of therapeutic agents in a releasable form. Releasable forms and compositions are designed to be sufficient to result in cellular uptake of the agent to provide a therapeutic effect.

Releasable forms include DILA2 amino acid compounds that bind and release the active agent. In some embodiments, release of the active agent can be provided by an acid labile linker.

Examples of acid labile linkers include those containing orthoester groups, hydrazone, cis-acetylmethyl, acetal, ketal, silyl ether, silazane, imine, citric anhydride, maleic anhydride, crown Linkers for ethers, azacrown ethers, thiacrown ethers, dithiobenzyl groups, cis-aconitic acid, cis-carboxyalkatriene, methacrylic acid, and mixtures thereof.

Examples of acid labile groups and linkers are found in US Pat.

Releasable forms of the compounds and compositions of the invention include molecules that bind the active agent and are capable of releasing a moiety that facilitates release of the agent. In some embodiments, the DILA2 amino acid compound may include a group that releases a small molecule such as ethanol that helps deliver the agent to the cell. The DILA2 amino acid compound can be bound to an active agent and subsequently contacted with a cell, or subsequently transported within a biological compartment with a local pH below physiological pH, being hydrolyzed in an acidic environment to release ethanol that aids in the delivery of the agent. In some embodiments, small molecules such as ethanol to aid in the delivery of the agent can be conjugated to the lipophilic component.

In some embodiments, the DILA2 amino acid compound can be mixed with a compound that releases a small molecule, such as ethanol, to help deliver the agent to the cell.

Releasable forms of the compounds and compositions of the present invention include DILA2 amino acid compounds that can be combined with an active agent and subsequently contacted with a cell, or subsequently transported within a biological compartment with a local pH below physiological pH, adjusted to The cationic form thus aids in the release of the reagents.

In some embodiments, the DILA2 amino acid compound can bind an active agent and can be mixed with a compound that can be adjusted to a cationic form in an acidic environment to aid in the release of the active agent.

Examples of hydrolyzable and adjustable groups are in U.S. Pat. Nos. 6,849,272; 6,200,599; and Z.H. Huang and F.C. Szoka, "Bioresponsive liposomes and their use formacromolecular delivery," given in .

In some embodiments, releasable forms of the compounds and compositions of the invention include a DILA2 amino acid compound that binds the active agent and can be combined with a lipid or compound that can be adjusted to a neutral form in an acidic environment to aid in the release of the active agent. mix. It can then be brought into an acidic environment for contact with cells, or subsequently transported within a biocompartment with a local pH below physiological pH.

Examples of compounds that are adjustable from anionic to neutral forms include cholesteryl succinate monoester (CHEMS), as described in US Pat. Nos. 6,897,196, 6,426,086, and 7,108,863. In some instances, CHEMS revealed pH-sensitive polymorphisms, as described in Cullis, 1463 Biochimica et Biophysica Acta 107-14 (2000).

In some embodiments, releasable forms of the compounds and compositions of the invention include a DILA2 amino acid compound capable of binding an active agent, which may be admixed with a pH-sensitive polymeric material.

Examples of pH sensitive polymeric materials are given in US Patent No. 6,835,393.

In some embodiments, release of the active agent can be provided by an enzymatically cleavable peptide.

In some aspects, the present invention provides a series of DILA2 amino acid compounds and salts thereof as shown in formula I:

R ³ -(C=O)-Xaa-ZR ⁴ Formula I

in

Xaa is any D- or L-amino acid residue of the general formula -NR ^N -CR ¹ R ² -(C=O)-, or a peptide of 2 to 20 amino acid residues, wherein

^R1 is a non-hydrogen, substituted or unsubstituted amino acid side chain;

^R is hydrogen or an organic group consisting of carbon, oxygen, nitrogen, sulfur and hydrogen atoms and having 1 to 20 carbon atoms or C(1-5)alkyl, cycloalkyl, cycloalkylalkyl, C (3-5) alkenyl, C (3-5) alkynyl, C (1-5) alkanoyl, C (1-5) alkanoyloxy, C (1-5) alkoxy, C (1 -5) Alkoxy-C(1-5) alkyl, C(1-5) alkoxy-C(1-5) alkoxy, C(1-5) alkyl-amino-C(1 -5) Alkyl-, C(1-5) dialkyl-amino-C(1-5) alkyl-, nitro-C(1-5) alkyl, cyano-C(1-5) Alkyl, aryl-C(1-5)alkyl, 4-biphenyl-C(1-5)alkyl, carboxyl or hydroxyl;

R ^N is hydrogen or an organic group consisting of carbon, oxygen, nitrogen, sulfur and hydrogen atoms and having 1 to 20 carbon atoms or C(1-5)alkyl, cycloalkyl, cycloalkylalkyl, C (3-5) alkenyl, C (3-5) alkynyl, C (1-5) alkanoyl, C (1-5) alkanoyloxy, C (1-5) alkoxy, C (1 -5) Alkoxy-C(1-5) alkyl, C(1-5) alkoxy-C(1-5) alkoxy, C(1-5) alkyl-amino-C(1 -5) Alkyl-, C(1-5) dialkyl-amino-C(1-5) alkyl-, nitro-C(1-5) alkyl, cyano-C(1-5) Alkyl, aryl-C(1-5)alkyl, 4-biphenyl-C(1-5)alkyl, carboxyl or hydroxyl;

^R is a lipophilic tail derived from a naturally occurring or synthetic phospholipid, glycolipid, triacylglycerol, glycerophospholipid, sphingolipid, ceramide, sphingomyelin, cerebroside, or ganglioside; or substituted or unsubstituted C (3-22) alkyl, C (6-12) cycloalkyl, C (6-12) cycloalkyl-C (3-22) alkyl, C (3-22) alkenyl, C ( 3-22) alkynyl, C (3-22) alkoxy or C (6-12) alkoxy-C (3-22) alkyl; or any other naturally occurring or synthetic lipid affinity A lipophilic tail or a lipophilic tail of any lipid described below, and may include a steroid;

^R is a lipophilic tail derived from naturally occurring or synthetic phospholipids, glycolipids, triacylglycerols, glycerophospholipids, sphingolipids, ceramides, sphingomyelin, cerebrosides, or gangliosides; or substituted or unsubstituted C (3-22) alkyl, C (6-12) cycloalkyl, C (6-12) cycloalkyl-C (3-22) alkyl, C (3-22) alkenyl, C ( 3-22) alkynyl, C (3-22) alkoxy or C (6-12) alkoxy-C (3-22) alkyl; or any other naturally occurring or synthetic lipid affinity A lipophilic tail or a lipophilic tail of any lipid described below, and may include a steroid;

Z is NH, O, S, _-CH2S- , _-CH2S (O)- or an organic linker consisting of 1 to 40 atoms selected from hydrogen, carbon, oxygen, nitrogen and sulfur atoms.

In some embodiments, R ³ is independently substituted or unsubstituted C(6-22) alkyl or C(6-22) alkenyl; R ⁴ is independently substituted or unsubstituted C(6-22) Alkyl or C(6-22)alkenyl.

Residue Xaa can be a D- or L-stereocenter.

In some embodiments, R ^is a non-hydrogen substituted or unsubstituted amino acid side chain, wherein the side chain substituent is an organic group consisting of 1 to 40 atoms selected from hydrogen, carbon, oxygen, nitrogen, and sulfur atoms.

In some embodiments, Z is an alkyl group or an organic linker synthetic polymer, such as a polyethylene glycol chain (PEG), or a PEG copolymer, such as PEG-polyurethane or PEG-polypropylene. See, eg, J. Milton Harris, Poly(ethylene glycol) chemistry: biotechnical and biomedical applications (1992).

In some embodiments, the present invention provides a series of DILA2 amino acid compounds as shown in formula I above, wherein:

Xaa is any D- or L-amino acid having the general formula -NR ^N -CR ¹ R ² -(C=O)-, wherein

R ¹ is a basic side chain of a non-hydrogen substituted or unsubstituted amino acid;

R ² is hydrogen, or C(1-5) alkyl,

R ^N is hydrogen, or C(1-5) alkyl,

^R is a lipophilic tail derived from a naturally occurring or synthetic phospholipid, glycolipid, triacylglycerol, glycerophospholipid, sphingolipid, ceramide, sphingomyelin, cerebroside, or ganglioside; or substituted or unsubstituted C (3-22) alkyl, C (6-12) cycloalkyl, C (6-12) cycloalkyl-C (3-22) alkyl, C (3-22) alkenyl, C ( 3-22) alkynyl, C (3-22) alkoxy or C (6-12) alkoxy-C (3-22) alkyl; or any other naturally occurring or synthetic lipid affinity A lipophilic tail, or a lipophilic tail of any of the lipids described below, and may include a steroid;

^R is a lipophilic tail derived from naturally occurring or synthetic phospholipids, glycolipids, triacylglycerols, glycerophospholipids, sphingolipids, ceramides, sphingomyelin, cerebrosides, or gangliosides; or substituted or unsubstituted C (3-22) alkyl, C (6-12) cycloalkyl, C (6-12) cycloalkyl-C (3-22) alkyl, C (3-22) alkenyl, C ( 3-22) alkynyl, C (3-22) alkoxy or C (6-12) alkoxy-C (3-22) alkyl; or any other naturally occurring or synthetic lipid affinity A lipophilic tail, or a lipophilic tail of any of the lipids described below, and may include a steroid;

Z is NH, O, S, _-CH2S- , _-CH2S (O)- or an organic linker consisting of 1 to 40 atoms selected from hydrogen, carbon, oxygen, nitrogen and sulfur atoms.

In some embodiments, the present invention provides a series of DILA2 amino acid compounds as shown in formula I above, wherein:

Xaa is any D- or L-amino acid having the general formula -NR ^N -CR ¹ R ² -(C=O)-, wherein

R ¹ is a basic side chain of a non-hydrogen substituted or unsubstituted amino acid;

R ² is hydrogen, or C(1-5) alkyl,

R ^N is hydrogen, or C(1-5) alkyl,

R ³ is substituted or unsubstituted C(3-22) alkyl, C(6-12) cycloalkyl, C(6-12) cycloalkyl-C(3-22) alkyl, C(3- 22) Alkenyl, C(3-22)alkynyl, C(3-22)alkoxy or C(6-12)alkoxy-C(3-22)alkyl;

R ⁴ is substituted or unsubstituted C(3-22) alkyl, C(6-12) cycloalkyl, C(6-12) cycloalkyl-C(3-22) alkyl, C(3- 22) Alkenyl, C(3-22)alkynyl, C(3-22)alkoxy or C(6-12)alkoxy-C(3-22)alkyl;

Z is NH, O, S, _-CH2S- , _-CH2S (O)- or an organic linker consisting of 1 to 40 atoms selected from hydrogen, carbon, oxygen, nitrogen and sulfur atoms.

In some embodiments, the present invention provides a series of DILA2 amino acid compounds as shown in formula I above, wherein:

Xaa is any D- or L-amino acid having the general formula -NR ^N -CR ¹ R ² -(C=O)-, wherein

R ¹ is a basic side chain of a non-hydrogen substituted or unsubstituted amino acid;

R ² is hydrogen, or C(1-5) alkyl,

R ^N is hydrogen, or C(1-5) alkyl,

R ³ is substituted or unsubstituted C(3-22) alkyl, C(6-12) cycloalkyl, C(6-12) cycloalkyl-C(3-22) alkyl, C(3- 22) Alkenyl, C(3-22)alkynyl, C(3-22)alkoxy or C(6-12)alkoxy-C(3-22)alkyl;

R ⁴ is substituted or unsubstituted C(3-22) alkyl, C(6-12) cycloalkyl, C(6-12) cycloalkyl-C(3-22) alkyl, C(3- 22) Alkenyl, C(3-22)alkynyl, C(3-22)alkoxy or C(6-12)alkoxy-C(3-22)alkyl;

Z is NH.

In some embodiments, the present invention provides a series of DILA2 amino acid compounds as shown in formula I above, wherein:

Xaa is any D- or L-amino acid having the general formula -NR ^N -CR ¹ R ² -(C=O)-, wherein

R ¹ is a basic side chain of a non-hydrogen substituted or unsubstituted amino acid;

R ² is hydrogen, or C(1-5) alkyl,

R ^N is hydrogen, or C(1-5) alkyl,

R ³ is substituted or unsubstituted C(3-22) alkyl, C(6-12) cycloalkyl, C(6-12) cycloalkyl-C(3-22) alkyl, C(3- 22) Alkenyl, C(3-22)alkynyl, C(3-22)alkoxy or C(6-12)alkoxy-C(3-22)alkyl;

R ⁴ is substituted or unsubstituted C(3-22) alkyl, C(6-12) cycloalkyl, C(6-12) cycloalkyl-C(3-22) alkyl, C(3- 22) Alkenyl, C(3-22)alkynyl, C(3-22)alkoxy or C(6-12)alkoxy-C(3-22)alkyl;

Z is O.

Cationic DILA2 amino acid compounds can be prepared in which, for example, Xaa has a basic side chain. Examples of amino acids with basic side chains include arginine (Arg), homoarginine (homoArg) (side chain-( _CH2 ) _4NH (C=NH) _NH2 ), nor-arginine (norArg )(side chain-(CH ₂ ) ₂ NH(C=NH)NH ₂ ), n-nor-arginine (n-Arg)(side chain-(CH ₂ )NH(C=NH)NH ₂ ), bird amino acid, lysine, homolysine, histidine, 1-methylhistidine, pyridylalanine (Pal), asparagine, N-ethylasparagine, glutamine and 4 -Aminophenylalanines and their side chain modified derivatives.

As used herein, the term "high", when referring to an amino acid, means an extra carbon is added to the side chain, while the term "normal", when referring to an amino acid, means a carbon is subtracted from the side chain. Thus, homolysine refers to the side chain -(CH ₂ ) ₅ NH ₂ .

Anionic DILA2 amino acid compounds can be prepared wherein, for example, Xaa is glutamic acid or aspartic acid.

Cationic and anionic DILA2 amino acid compounds can also be prepared in which the amino acid side chain contains ionizing groups or substituents.

Non-cationic DILA2 amino acid compounds can be prepared wherein, for example, Xaa is leucine, valine, alanine or serine.

In some embodiments, Xaa is NG ^- methylarginine, symmetric or asymmetric ^NG , ^NG -dimethylarginine, ^NG -methyl-homoarginine, symmetric or Asymmetric ^NG , NG ^- dimethyl-homoarginine, NG ^- methyl-norarginine, symmetrical or asymmetrical ^NG , ^NG -dimethyl-norarginine or NG ^- methyl-nor-nor-arginine, symmetrical or asymmetrical ^NG , ^NG -dimethyl-nor-nor-arginine.

In some embodiments, Xaa is NG ^- ethylarginine, symmetric or asymmetric ^NG , ^NG -diethylarginine, ^NG -ethyl-homoarginine, symmetric or Asymmetric ^NG , NG- ^diethyl -homoarginine, NG ^- ethyl-norarginine, symmetric or asymmetric ^NG , ^NG -diethyl-norarginine or NG ^-ethyl -nor-nor-arginine, symmetrical or asymmetrical ^NG , NG ^-diethyl -nor-nor-arginine.

In some embodiments, Xaa is NG ^- alkylarginine, symmetric or asymmetric ^NG , ^NG -dialkylarginine, NG ^- alkyl-homoarginine, symmetric or Asymmetric ^NG , NG ^- dialkyl-homoarginine, NG ^- alkyl-norarginine, symmetric or asymmetric ^NG , ^NG -dialkyl-norarginine or NG ^- alkyl-n-n-arginine, symmetric or asymmetric ^NG , ^NG -dialkyl-n-n-arginine.

In some embodiments, Xaa is an amino acid with a guanidine- or amidine-containing side chain. For example, the side chain of an Xaa residue can include, for example, guanidino, amidino, dihydroimidazole, 4-guanidino-phenyl, 4-amidino-phenyl, N-amidino-piperidine, N-amidino- A group of piperazine, 4,5-dihydroimidazole, 2-(N-amidino)-pyrrolidinyl or 4-[(2-aminopyrimidinyl)]ethyl.

Examples of Xaa side chains include the following structures, and salt forms thereof:

Examples of substituted side chains of amino acids suitable for releasable forms of DILA2 amino acid compounds include releasing functional groups with a pKa of about 5 to about 7.5 or about 6 to about 7. In general, the releasing functional groups of weak bases can exhibit a predominantly neutral form at local pHs above the pKa and a predominantly ionic form at local pHs below the pKa. The releasing functional group of a weak acid can assume an ionic form at a local pH above the pKa, and a neutral form at a local pH below the pKa. See, eg, P. Heinrich Stahl, Handbook of Pharmaceutical Salts, (2002).

In some embodiments, Xaa may have a side chain containing a functional group with a pKa of 5 to 7.5.

Examples of substituted side chains of amino acids suitable for releasable forms of DILA2 amino acid compounds include 1-methylhistidine.

Examples of substituted side chains of amino acids suitable for releasable forms of DILA2 amino acid compounds include 3,5-diiodotyrosine.

Examples of substituted side chains of amino acids suitable for releasable forms of DILA2 amino acid compounds include the following structures:

Examples of DILA2 amino acid compounds include the structure:

Examples of substituents on the amino acid side chains of DILA2 amino acid compounds suitable for releasable form include releasable functional groups derived from: 3,5-diiodo-tyrosine, 1-methylhistidine, 2-methyl Butyric acid, 2-o-anisylpropionic acid, meso-tartaric acid, 4,6-dimethylpyrimidinamine, terephthalic acid, creatinine, butyric acid, N,N-dimethyl-1-naphthalene Amine, valeric acid, 4-methylvaleric acid, N-methylaniline, 1,10-phenanthroline, 3-pyridinecarboxylic acid, hexanoic acid, propionic acid, 4-aminobenzoic acid, 2-methyl Propionic acid, heptanoic acid, octanoic acid, cyclohexanecarboxylic acid, quinoline, 3-aminoquinoline, 2-aminobenzoic acid, 4-pyridinecarboxylic acid, nonanoic acid, melamine, 8-hydroxyquinoline, trimethylacetic acid , 6-methoxyquinoline, 4-(methylamino)benzoic acid, p-methylaniline, 3-(methylamino)benzoic acid, malic acid, N-ethylaniline, 2-benzylpyridine, 3,6-dinitrophenol, N,N-dimethylaniline, 2,5-dimethylpiperazine, p-aminophenetole, 5-methylquinoline, 2-phenylbenzimidazole, pyridine, Picolinic acid, 3,5-diiodotyrosine, p-anisidine, 2-(methylamino)benzoic acid, 2-aminothiazole, glutaric acid, adipic acid, isoquinoline, itaconic acid, phthalic acid Dicarboxylic acid, benzimidazole, piperazine, pimelic acid, acridine, phenanthridine, succinic acid, methylsuccinic acid, 4-methylquinoline, 3-picoline, 7-hydroxyisoquinoline, propane Diacid, methylmalonic acid, 2-methylquinoline, 2-ethylpyridine, 2-picoline, 4-picoline, histamine, histidine, maleic acid, cis-1, 2-cyclohexanediamine, 3,5-lutidine, 2-ethylbenzimidazole, 2-methylbenzimidazole, cacodylic acid, perimidine, citric acid, isocitric acid , 2,5-lutidine, papaverine, 6-hydroxy-4-methylpteridine, L-thyroxine, 3,4-lutidine, methoxypyridine, trans-1,2- Cyclohexanediamine, 2,5-pyridinediamine, l-1-methylhistidine, l-3-methylhistidine, 2,3-lutidine, xantterin, 1,2 -Propanediamine, N,N-diethylaniline, alluric acid, 2,6-lutidine, L-carnosine, 2-aminopyridine, N-b-alanyl histidine, pilocarpine, 1-methylpyridine Imidazole, 1H-imidazole, 2,4-lutidine, 4-nitrophenol, 2-nitrophenol, tyrosinamide, 5-hydroxyquinazoline, 1,1-cyclopropanedicarboxylic acid, 2 , 4,6-Collidine, Verona, 2,3-Dichlorophenol, 1,2-Ethylenediamine, 1-Aminoisoquinoline and combinations thereof.

In some embodiments, the structure of a series of DILA2 amino acid compounds corresponding to Formula I is as follows:

wherein R ¹ , R ² , R ^N , R ³ and R ⁴ are as defined above.

In some embodiments, ^R3 and ^R4 are independently selected lipophilic tails that confer sufficient lipophilic character or lipophilicity, such as separated by water/octanol, for delivery across membranes or uptake by cells. These tails provide amphiphilic molecules when used in DILA2 amino acid compounds. The lipophilic tail may be derived, inter alia, from phospholipids, glycolipids, triacylglycerols, glycerophospholipids, sphingolipids, ceramides, sphingomyelins, cerebrosides or gangliosides, etc., and may include steroids.

In some embodiments, ^R3 and ^R4 can independently be a lipophilic tail with a glycerol backbone.

In some embodiments, R ^and R can be ^{independently} C10 alkyl, C11 alkyl, C12 alkyl, C13 alkyl, C14 alkyl, C15 alkyl, C16 alkyl, C17 alkyl, C18 alkyl , C19 alkyl, C20 alkyl, C21 alkyl or C22 alkyl.

In some embodiments, R ^{and R} can independently be a lipophilic tail having one of the following structures:

In the above structures, X represents the tail atom directly connected to the terminal of the amino acid residue, and is designated as one atom thereof with a numerical designation, for example "18:3". In some embodiments, X can be a carbon, nitrogen or oxygen atom.

In some embodiments, R ^{and R} can independently be a lipophilic tail having one of the following structures:

wherein X is as defined above.

In some embodiments, R ^{and R} are independently selected lipophilic tails, which may include cholesterol, sterols, or steroids, such as sterane, estane, androstane, pregnane, cholane, cholestane, ergot Steranes, campesteranes, porostanes, stigmasteranes, gorgostanes, lanostanes, cyclopolanes, and any of the aforementioned sterol or zoosterol derivatives and their biological intermediates and Precursors may include, for example, cholesterol, lanosterol, stigmasterol, dihydrolanosterol, zymosterol, zymostanol, streptosterol, 7-dehydrocholesterol and their mixtures and derivatives.

In some embodiments, R ^{and R} can be independently derived from a fatty acid-like tail, such as derived from myristic acid (C14:0) alkenyl, palmitic acid (C16:0) alkenyl, stearic acid (C18:0) alkenyl, 0) Alkenyl, oleic acid (C18:1, double bond at carbon 9) alkenyl, linoleic acid (C18:2, carbon double bond at carbon 9 or 12) alkenyl, linolenic acid (C18:3, double bond At carbon 9, 12 or 15) alkenyl, arachidonic acid (C20:4, double bond at carbon 5, 8, 11 or 14) alkenyl and eicosapentaenoic acid (C20:5, double bond at carbon 5, 8, 11, 14 or 17) alkenyl tail. For other examples of fatty acid-like tails see Donald Voet and Judith Voet, Biochemistry, 3rd Edition (2005), p.383.

In some embodiments, R ^{and R} can be independently derived from isoprenoids.

As used herein, the term "amino acid" includes naturally occurring and non-naturally occurring amino acids. Thus DILA2 amino acid compounds can be prepared from genetically encoded amino acids, naturally occurring non-genetically encoded amino acids, or synthetic amino acids.

Examples of amino acids include Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.

Examples of amino acids include azetidine, 2-aminooctadecanoic acid, 2-aminoadipic acid, 3-aminoadipic acid, 2,3-diaminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid , 2,3-diaminobutyric acid, 2,4-diaminobutyric acid, 2-aminoisobutyric acid, 4-aminoisobutyric acid, 2-aminopimelic acid, 2,2'-diaminopimelic acid , 6-aminocaproic acid, 6-aminocaproic acid, 2-aminoheptanoic acid, desmosin, ornithine, citrulline, N-methylisoleucine, norleucine, tertiary leucine, benzene Glycine, tert-butylglycine, N-methylglycine, sarcosine, N-ethylglycine, cyclohexylglycine, 4-oxo-cyclohexylglycine, N-ethylasparagine, cyclohexylalanine, tert-butylalanine, naphthylalanine, pyridylalanine, 3-chloroalanine, 3-benzothienylalanine, 4-halophenylalanine, 4-chlorophenylalanine amino acid, 2-fluorophenylalanine, 3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine, 2-thienylalanine, methionine, methionine sulfoxide, homoarginine, N-arginine, n-nor-arginine, N-acetyllysine, 4-aminophenylalanine, N-methylvaline, homocysteine, homoserine, hydroxylysine, Allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, allo-isoleucine, 6-N-methyllysine, norvaline, O-ene Propyl-serine, O-allyl-threonine, alpha-aminocaproic acid, alpha-aminovaleric acid and pyroglutamic acid.

As used herein, the term "amino acid" includes α- and β-amino acids.

Other amino acid residues can be found in Fasman, CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Inc. (1989).

In general, compounds may include one or more chiral centers. Compounds containing one or more chiral centers may include compounds known as "isomers", "stereoisomers", "diastereoisomers", "enantiomers", "optical isomers" body" or "racemic mixture". Stereochemical nomenclature conventions, such as Cahn, Ingold and Prelog's stereoisomer nomenclature, and methods for determining stereochemistry and separating stereoisomers are known in the art. See, eg, Michael B. Smith and Jerry March, March's Advanced Organic Chemistry, 5th ed., 2001. The compounds and structures of the present invention are intended to include all possible isomers, stereoisomers, diastereomers, enantiomers and/or optical isomers, which are understood to mean that with a given compound or structure, Including any mixture, racemate or other forms thereof.

Examples of DILA2 amino acid compounds include ^R3- (C=O)-Arg-NH- ^R4 , wherein Arg is D- or L-arginine, and ^R3 and ^R4 are independently alkyl or alkenyl.

Examples of DILA2 amino acid compounds include the following structures:

Examples of DILA2 amino acid compounds include the following structures:

Examples of DILA2 amino acid compounds include the following structures:

Examples of DILA2 amino acid compounds include R ³ -(C=O)-n-Arg-NH-R ⁴ , wherein n-Arg is D- or L-n-arginine, and R ³ and R ⁴ are independently alkyl or alkene base.

Examples of DILA2 amino acid compounds include the following structures:

Examples of DILA2 amino acid compounds include R3-(C=O)-n-n-Arg-NH- ^R4 , wherein n-n-Arg is D- or L-n-n-arginine, and ^R3 and ^R4 are independently alkyl radicals such as heptyl, octyl, nonyl, decyl and undecyl.

Examples of DILA2 amino acid compounds include the following structures:

Examples of DILA2 amino acid compounds include R ³ -(C=O)-homoArg-NH-R ⁴ , wherein homoArg is D- or L-homoarginine, and R ³ and R ⁴ are independently alkyl, such as Heptyl, Octyl, Nonyl, Decyl and Undecyl.

Examples of DILA2 amino acid compounds include R ³ -(C=O)-4-pyridylalanine-NH-R ⁴ , wherein pyridylalanine is D- or L-pyridylalanine, and R ³ and ^R4 is independently alkyl, such as heptyl, octyl, nonyl, decyl and undecyl. Examples of DILA2 amino acid compound R ³ -(C═O)-pyridylalanine-NH-R ⁴ include pharmaceutically acceptable pyridinium salts such as 4-[N-methylpyridyl]alanine chloride. Examples of pyridylalanine DILA2 amino acid compounds include the following structures:

Examples of DILA2 amino acid compounds include R ³ -(C═O)-Lys-NH-R ⁴ , wherein R ³ and R ⁴ are independently alkyl or alkenyl.

Examples of DILA2 amino acid compounds include the following structures:

Examples of DILA2 amino acid compounds include R ³ -(C═O)-His-NH-R ⁴ , wherein R ³ and R ⁴ are independently alkyl or alkenyl. Examples of HisDILA2 amino acid compounds include the following structures:

Examples of DILA2 amino acid compounds include R ³ -(C═O)-Xaa-OR ⁴ , wherein R ³ is alkyl and R ⁴ is sphingosine.

Examples of DILA2 amino acid compounds include the following structures:

Examples of DILA2 amino acid compounds include R ³ -(C═O)-Xaa-NH-R ⁴ , wherein R ³ and R ⁴ are alkyl or alkenyl. Examples of DILA2 amino acid compounds include the following structures:

Examples of DILA2 amino acid compounds include the following structures:

Examples of DILA2 amino acid compounds include the following structures:

Examples of DILA2 amino acid compounds include the following structures:

C _18:1 -Ser(succinylated)-C ₁₆

Examples of DILA2 amino acid compounds include (C10 acyl)-Arg-NH-(C10 alkyl) (SEQ ID NO: 11), (C12 acyl)-Arg-NH-(C12 alkyl) (SEQ ID NO: 11), ( C14 acyl)-Arg-NH-(C14 alkyl) (SEQ ID NO: 11), (C16 acyl)-Arg-NH-(C16 alkyl) (SEQ ID NO: 11), (C18 acyl)-Arg-NH -(C18Alkyl)(SEQ ID NO: 11), (C10Acyl)-HomoArg-NH-(C10Alkyl), (C12Acyl)-HomoArg-NH-(C12Alkyl), (C14Acyl) -Homo Arg-NH-(C14 Alkyl), (C16 Acyl)-Homo Arg-NH-(C16 Alkyl), (C18 Acyl)-Homo Arg-NH-(C18 Alkyl), (C10 Acyl)-Nor Arg-NH-(C10 alkyl), (C12 acyl)-n-Arg-NH-(C12 alkyl), (C14 acyl)-n-Arg-NH-(C14 alkyl), (C16 acyl)-n-Arg- NH-(C16 alkyl), (C18 acyl)-n-Arg-NH-(C18 alkyl), (C10 acyl)-n-n-Arg-NH-(C10 alkyl), (C12 acyl)-n-n-Arg -NH-(C12 alkyl), (C14 acyl)-n-n-Arg-NH-(C14 alkyl), (C16 acyl)-n-n-Arg-NH-(C16 acyl), (C18 acyl)-n-n Arg-NH-(C18 alkyl), (C10 acyl)-4-Pal-NH-(C10 alkyl), (C12 acyl)-4-Pal-NH-(C12 acyl), (C14 acyl)-4 -Pal-NH-(C14 alkyl), (C16 acyl)-4-Pal-NH-(C16 alkyl), (C18 acyl)-4-Pal-NH-(C18 alkyl), (C10 acyl)- 4-Pal(Me)-NH-(C10 alkyl), (C12 acyl)-4-Pal(Me)-NH-(C12 alkyl), (C14 acyl)-4-Pal(Me)-NH-( C14 alkyl), (C16 acyl)-4-Pal(Me)-NH-(C16 alkyl) and (C18 acyl)-4-Pal(Me)-NH-(C18 alkyl).

In general, the designation "C14-n-Arg-C14" refers, for example, to (C13 alkyl)-(C=O)-n-Arg-NH-(C14 alkyl), which is equivalent to (C14 acyl)-n-Arg-NH- (C14 alkyl).

Examples of DILA2 amino acid compounds include (C10 acyl)-D-Arg-L-Arg-NH-(C10 alkyl), (C12 acyl)-D-Arg-L-Arg-NH-(C12 alkyl), (C14 Acyl)-D-Arg-L-Arg-NH-(C14Alkyl), (C16Acyl)-D-Arg-L-Arg-NH-(C16Alkyl), (C18Acyl)-D-Arg-L -Arg-NH-(C18 alkyl), (C10 acyl)-D-homoArg-L-homoArg-NH-(C10 alkyl), (C12 acyl)-D-homoArg-L-homoArg-NH -(C12 alkyl), (C14 acyl)-D-homoArg-L-homoArg-NH-(C14 alkyl), (C16 acyl)-D-homoArg-L-homoArg-NH-(C16 alkane base), (C18 acyl)-D-homoArg-L-homoArg-NH-(C18 alkyl), (C10 acyl)-D-n-Arg-L-n-Arg-NH-(C10 alkyl), ( C12 acyl)-D-n-Arg-L-n-Arg-NH-(C12 acyl), (C14 acyl)-D-n-Arg-L-n-Arg-NH-(C14 acyl), (C16 acyl)- D-n-Arg-L-n-Arg-NH-(C16 alkyl), (C18 acyl)-D-n-Arg-L-n-Arg-NH-(C18 alkyl), (C10 acyl)-D-n- n-Arg-L-n-n-Arg-NH-(C10 alkyl), (C12 acyl)-D-n-n-Arg-L-n-n-Arg-NH-(C12 alkyl), (C14 acyl)-D-n n-Arg-L-n-n-Arg-NH-(C14 alkyl), (C16 acyl)-D-n-n-Arg-L-n-n-Arg-NH-(C16 alkyl), (C18 acyl)-D -n-n-Arg-L-n-n-Arg-NH-(C18 alkyl).

Examples of DILA2 amino acid compounds include (C10 acyl)-His-Arg-NH-(C10 acyl), (C12 acyl)-His-Arg-NH-(C12 acyl), (C14 acyl)-His-Arg-NH -(C14Alkyl), (C16Acyl)-His-Arg-NH-(C16Alkyl), (C18Acyl)-His-Arg-NH-(C18Alkyl), (C10Acyl)-His-Arg- NH-(C10 alkyl), (C12 acyl)-His-Arg-NH-(C12 acyl), (C14 acyl)-His-Arg-NH-(C14 acyl), (C16 acyl)-His-Arg -NH-(C16 alkyl), (C18 acyl)-His-Arg-NH-(C18 acyl), (C10 acyl)-His-Arg-(C10 acyl), (C12 acyl)-His-Arg- NH-(C12 alkyl), (C14 acyl)-His-Arg-NH-(C14 acyl), (C16 acyl)-His-Arg-NH-(C16 acyl), (C18 acyl)-His-Arg -NH-(C18 alkyl), (C10 acyl)-His-Arg-NH-(C10 acyl), (C12 acyl)-His-Arg-NH-(C12 acyl), (C14 acyl)-His- Arg-NH-(C14 alkyl), (C16 acyl)-His-Arg-NH-(C16 alkyl), (C18 acyl)-His-Arg-NH-(C18 alkyl).

Examples of DILA2 amino acid compounds include (C10 acyl)-His-Asp-NH-(C10 acyl), (C12 acyl)-His-Asp-NH-(C12 acyl), (C14 acyl)-His-Asp-NH -(C14 alkyl), (C16 acyl)-His-Asp-NH-(C16 acyl), (C18 acyl)-His-Asp-NH-(C18 acyl), (C10 acyl)-His-Asp- NH-(C10 alkyl), (C12 acyl)-His-Asp-NH-(C12 acyl), (C14 acyl)-His-Asp-NH-(C14 acyl), (C16 acyl)-His-Asp -NH-(C16 alkyl), (C18 acyl)-His-Asp-NH-(C18 acyl), (C10 acyl)-His-Asp-(C10 acyl), (C12 acyl)-His-Asp- NH-(C12 alkyl), (C14 acyl)-His-Asp-NH-(C14 acyl), (C16 acyl)-His-Asp-NH-(C16 acyl), (C18 acyl)-His-Asp -NH-(C18 alkyl), (C10 acyl)-His-Asp-NH-(C10 acyl), (C12 acyl)-His-Asp-NH-(C12 acyl), (C14 acyl)-His- Asp-NH-(C14 alkyl), (C16 acyl)-His-Asp-NH-(C16 alkyl), (C18 acyl)-His-Asp-NH-(C18 alkyl).

Examples of DILA2 amino acid compounds include (C10 acyl)-Pal-Arg-NH-(C10 acyl), (C12 acyl)-Pal-Arg-NH-(C12 acyl), (C14 acyl)-Pal-Arg-NH -(C14Alkyl), (C16Acyl)-Pal-Arg-NH-(C16Alkyl), (C18Acyl)-Pal-Arg-NH-(C18Alkyl), (C10Acyl)-Pal-Arg- NH-(C10 alkyl), (C12 acyl)-Pal-Arg-NH-(C12 acyl), (C14 acyl)-Pal-Arg-NH-(C14 acyl), (C16 acyl)-Pal-Arg -NH-(C16Alkyl), (C18Acyl)-Pal-Arg-NH-(C18Alkyl), (C10Acyl)-Pal-Arg-(C10Alkyl), (C12Acyl)-Pal-Arg- NH-(C12Alkyl), (C14Acyl)-Pal-Arg-NH-(C14Alkyl), (C16Acyl)-Pal-Arg-NH-(C16Alkyl), (C18Acyl)-Pal-Arg -NH-(C18 alkyl), (C10 acyl)-Pal-Arg-NH-(C10 acyl), (C12 acyl)-Pal-Arg-NH-(C12 acyl), (C14 acyl)-Pal- Arg-NH-(C14 alkyl), (C16 acyl)-Pal-Arg-NH-(C16 alkyl), (C18 acyl)-Pal-Arg-NH-(C18 alkyl).

DILA2 amino acid compounds can be prepared as polymers or multimers, such as dimers, trimers or tetramers. Polymers or multimers can be prepared from one or more than one DILA2 amino acid compound. In some embodiments, polymeric or multimeric DILA2 amino acid compounds can be synthesized by providing thiols or other crosslinkable groups on the amino acid side chains or using linked or tethered amino acid structures (such as desmosin or citrulline). amino acid) to prepare. In other embodiments, polymeric or multimeric DILA2 amino acid compounds can be prepared using bioconjugate linker chemistry.

Examples of DILA2 amino acid compounds include the following structures:

DILA2 amino acid compounds can be prepared as conjugates with peptide or polymer chains covalently linked to amino acid side chains. Peptide or polymer chains can be linked using amino acid side chain reactive groups, such as the thiol or methylthiol groups of cysteine or methionine, respectively, or the alcohol group of serine, or the amino group of lysine. Peptide or polymer chains can be linked using any reactive group of substituted or modified amino acid side chains. Various linker groups such as NHS, maleimide groups and bioconjugation techniques and linkers can be used.

DILA2 amino acid compounds can be prepared as constructs linked to oligomeric or polymeric frameworks. For example, the DILA2 amino acid compound can be linked to polyethylene glycol, polypropylene glycol, oligonucleotide network or lattice, poly(amino acid), carbohydrate, dextran, hydrogel, or starch.

DILA2 amino acid compounds can be prepared as constructs linked to pharmaceutical compounds or compositions or biologically active agents. For example, DILA2 amino acid compounds can be linked to nucleic acid drugs such as regulatory or interfering RNA.

Examples of DILA2 amino acid compounds include the following structures:

where R is any amino acid side chain.

The compounds and compositions of the invention may incorporate solubilizing or functionalizing groups or structures including polymeric structures. See, e.g., R.L. Dunn and R.M. Ottenbrite, Polymeric Drugs and Drug Delivery Systems, ACS Symp. Ser. 469 (1991). DILA2 amino acid compounds can be derivatized to improve solubility, such as linking diols, preparing quaternary ammonium or charged groups, linking hydroxyl or amine groups such as alcohols, polyols or polyethers, or linking polyethyleneimine, polyethylene glycol alcohol or polypropylene glycol. The molecular weight of the attached polymeric component such as polyethylene glycol can be any value, for example 200, 300, 400, 500, 750, 1000, 1250, 1500, 2000, 3000, 4000, 5000, 7500, 10,000, 15,000, 20,000 , 25,000 or 30,000 Da or larger. For example, polyethylene glycol chains may be linked through amino or other reactive groups of amino acid side chains.

In general, herein, and unless otherwise indicated, conventional chemical terms refer to all groups of a given type, including groups having any number and type of atoms. For example "alkenyl" broadly refers to an alkyl group having 2 to 22 carbon atoms as defined below, wherein (C18:1)alkenyl refers to an alkenyl group having 18 carbon atoms and 1 double bond.

As used herein, the term "alkyl" refers to a saturated, branched or unbranched, substituted or unsubstituted aliphatic group containing 1 to 22 carbon atoms. These definitions may apply to the alkyl portion of other groups such as alkoxy, alkanoyl, aralkyl and other groups defined below. As used herein, the term "cycloalkyl" refers to a saturated, substituted or unsubstituted cycloalkyl ring containing 3 to 12 carbon atoms.

As used herein, the term "alkenyl" refers to an unsaturated, branched or unbranched, substituted or unsubstituted alkyl or cycloalkane having 2 to 22 carbon atoms and at least 1 carbon-carbon double bond base. As used herein, the term "alkynyl" refers to an unsaturated, branched or unbranched, substituted or unsubstituted alkyl or cycloalkane having 2 to 22 carbon atoms and at least 1 carbon-carbon triple bond base.

As used herein, the term "alkoxy" refers to an alkyl, cycloalkyl, alkenyl or alkynyl group covalently bonded to an oxygen atom. As used herein, the term "alkanoyl" refers to a -C(=O)-alkyl group, which may also be referred to as "acyl". As used herein, the term "alkanoyloxy" refers to -O-C(=O)-alkyl. As used herein, the term "alkylamino" refers to the group -NRR', wherein R and R' are each hydrogen or alkyl, and at least one of R and R' is alkyl. Alkylamino includes groups such as piperidinyl, wherein R and R' form a ring. The term "alkylaminoalkyl" refers to -alkyl-NRR'.

As used herein, the term "aryl" refers to any stable monocyclic, bicyclic or polycyclic carbocyclic ring system having 4 to 12 atoms in each ring, wherein at least one ring is aromatic. Some examples of aryl groups include phenyl, naphthyl, tetrahydronaphthyl, indanyl, and biphenyl. Where the aryl substituent is bicyclic and one ring is non-aromatic, it is understood that the aromatic ring is attached. Aryl groups can be substituted or unsubstituted.

As used herein, the term "heteroaryl" refers to any stable monocyclic, bicyclic or polycyclic carbocyclic ring system of 4 to 12 atoms in each ring, wherein at least one ring is aromatic and contains 1 to 4 heteroatoms selected from oxygen, nitrogen and sulfur. Some examples of heteroaryl groups include acridinyl, quinoxalinyl, pyrazolyl, indolyl, benzotriazolyl, furyl, thienyl, benzothienyl, benzofuryl, quinolinyl, isoquinolinyl, Azolyl, iso Azolyl, pyrazinyl, pyridazinyl, pyridyl, pyrimidinyl, pyrrolyl and tetrahydroquinolinyl. Heteroaryl includes N-oxide derivatives of nitrogen-containing heteroaryl groups.

As used herein, the term "heterocycle" or "heterocyclyl" refers to an aromatic or non-aromatic ring system of 5 to 22 atoms, of which 1 to 4 ring atoms are heteroatoms selected from oxygen, nitrogen and sulfur . Thus, the heterocycle may be heteroaryl or its dihydro or tetrahydro forms.

As used herein, the term "aroyl" refers to an aryl group derived from an aromatic carboxylic acid, such as a substituted benzoic acid. The term "aralkyl" refers herein to an aryl group bonded to an alkyl group, such as phenyl.

As used herein, the term "carboxy" means a group of formula -C(=O)OH or -C(=O) ^O- . The terms "carbonyl" and "acyl" refer herein to a group in which an oxygen atom is double bonded to a carbon atom >C=O. As used herein, the term "hydroxyl" refers to -OH or ^-O- . As used herein, the term "nitrile" or "cyano" refers to -CN. The term "halogen" or "halo" refers to fluoro (-F), chloro (-Cl), bromo (-Br) and iodo (-I).

As used herein, the term "substituted" means that an atom has one or more substitutions or substituents, which may be the same or different, and may include hydrogen substituents. Thus, the terms alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, alkanoyl, alkanoyloxy, alkylamino, alkylaminoalkyl, aryl, heteroaryl, heterocycle, aroyl And aralkyl refers herein to groups including substituted variants. Substituted variants include linear, branched and cyclic variants, and groups having substituents replacing one or more hydrogens attached to any carbon atom of the group. Substituents which may be attached to a carbon atom of a group include alkyl, cycloalkyl, alkenyl, alkynyl, alkoxy, alkanoyl, alkanoyloxy, alkylamino, alkylaminoalkyl, aryl, Heteroaryl, heterocycle, aroyl, aralkyl, acyl, hydroxy, cyano, halo, haloalkyl, amino, aminoacyl, alkylaminoacyl, acyloxy, aryloxy, aryloxyalkyl , mercapto, nitro, carbamoyl, carbamoyl and heterocycle. For example, the term ethyl includes _, but is not limited to _{: -CH2CH3} , _-CHFCH3 , _-CF2CH3 , _-CHFCH2F , _-CHFCHF2 , _{-CHFCF3 , -CF2CH2F} , _{-CF2CHF 2. -CF2CF3} and other variants as described above _. In general, a substituent can be further substituted with any atom or group of atoms.

DILA2 amino acid compounds can be synthesized by methods known in the art.

Methods for preparing various organic groups and protecting groups are known in the art, and their use and modification are generally within the purview of those skilled in the art. See, e.g., Stanley R. Sandler and WolfKaro, Organic Functional Group Preparations (1989); Greg T. Hermanson, Bioconjugate Techniques (1996); Leroy G. Wade, Compendium Of Organic Synthetic Methods (1980); Examples of protecting groups are found in T.W. Greene and P.G.M. Wuts, Protective Groups In Organic Synthesis (3rd ed., 1991).

For example, the DILA2 amino acid compound PONA can be synthesized according to the following route:

A pharmaceutically acceptable salt of a peptide or protein composition of the invention that is sufficiently basic may be an acid addition salt with, for example, an inorganic or organic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid , nitric acid, phosphoric acid, chlorosulfonic acid, trifluoroacetic acid, citric acid, maleic acid, acetic acid, propionic acid, oxalic acid, malic acid, maleic acid, malonic acid, fumaric acid or tartaric acid, and alkane sulfonic acids or aromatic hydrocarbons Sulfonic acids such as methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, chlorobenzenesulfonic acid, toluenesulfonic acid, naphthalenesulfonic acid, naphthalene disulfonic acid and camphorsulfonic acid.

A pharmaceutically acceptable salt of a peptide or protein composition of the invention that is sufficiently acidic may be an alkali metal salt, such as sodium or potassium, or an alkaline earth metal salt, such as calcium or magnesium, or zinc or manganese, or ammonium salts, or salts of organic bases that can provide physiologically acceptable cations, such as methylamine, dimethylamine, trimethylamine, triethylamine, ethanolamine, diethanolamine, triethanolamine, ethylenediamine, triethanolamine, N-methylamine Salts of glucosamine, piperidine, morpholine or tris-(2-hydroxyethyl)amine, and include salts of amino acids such as arginine, and salts of organic acids such as glucuronic acid or galacturonic acid. See, eg, Berge et al., J. Pharm. Sci. 66:1-19,1977.

The salt or pharmaceutically acceptable salt of the composition of the present invention comprising interfering RNA agent and DILA2 amino acid compound, lipid, peptide or protein may also comprise a salt complex of interfering RNA agent and DILA2 amino acid compound, lipid, peptide or protein . The salt complex of the interfering RNA agent and the DILA2 amino acid compound, lipid, peptide or protein can be formed from a pharmaceutically acceptable salt of the interfering RNA agent, or from a pharmaceutically acceptable salt of the DILA2 amino acid compound, lipid, peptide or protein salt formation.

Certain compounds of the present invention may contain both basic and acidic functionalities, which enable the compounds to be prepared as base or acid addition salts.

Some of the compounds, peptides and/or protein compositions of the invention may possess one or more chiral centers and/or geometric isomeric centers (E- and Z-isomers), and it is understood that the invention encompasses all such optical centers Isomers, diastereomers, geometric isomers and mixtures thereof.

The present invention encompasses any and all tautomers, solvated or unsolvated, hydrated or unhydrated forms, and any atomic isotopic forms of the compounds, peptide and/or protein compositions disclosed herein.

Lipid

In some aspects of the invention, one or more DILA2 amino acid compounds and one or more lipids can be used to deliver and administer regulatory RNA components, RNA antagonists, interfering RNA or nucleic acids. More specifically, compositions of the invention may include one or more DILA2 amino acid compounds as well as cationic and non-cationic lipids.

Cationic lipids can be monocationic or polycationic. Some cationic lipids include neutral lipids and lipids that have approximately zero net charge at a particular pH, eg, amphoteric lipids. Non-cationic lipids also include anionic lipids.

In some embodiments, the composition is a mixture or complex of an RNA component with a DILA2 amino acid compound and a cationic lipid. In some embodiments, the composition can be a mixture or complex of one or more regulatory or interfering RNA agents with one or more DILA2 amino acid compounds and one or more cationic lipids.

The compounds and compositions of the present invention may be mixed or linked with various targeting ligands or agents for delivery of active agents to cells, tissues, organs or regions of an organism. Examples of targeting agents include antibodies, ligands for receptors, peptides, proteins, lectins, (poly)saccharides, galactose, mannose, cyclodextrins, nucleic acids, DNA, RNA, nucleic acid aptamers and polyamino acids.

Examples of cationic lipids include N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA); 1,2-bis(oleoyl) Oxy)-3-3-(trimethylammonium)propane (DOTAP), 1,2-bis(dimyristyloxy)-3-3-(trimethylammonium)propane (DMTAP); 1,2 -Dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE); Dimethyldioctadecylammonium bromide (DDAB); 3-(N-(N',N' -Dimethylaminoethane)carbamoyl)cholesterol (DC-Chol); 3β-[N',N'-biguanidinoethyl-aminoethane]carbamoylcholesterol (BGTC); 2-(2 -(3-(bis(3-aminopropyl)amino)propylamino)acetamido)-N,N-ditetradecylacetamide (RPR209120); their pharmaceutically acceptable salts and their mixture.

Examples of cationic lipids include 1,2-dienoyl-tin-glycero-3-ethylphosphocholine (EPC), such as 1,2-dioleoyl-tin-glycero-3-ethylphosphocholine Base, 1,2-distearoyl-tin-glyceryl-3-ethylphosphocholine, 1,2-dipalmitoyl-tin-glyceryl-3-ethylphosphocholine, their pharmaceutically acceptable Accepted salts and mixtures thereof.

Examples of cationic lipids include 1,2-distearoyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleoyloxy-N,N-dimethyl -3-aminopropane (DODMA), 1,2-dioleoyloxy-N,N-dimethyl-3-aminopropane (DLinDMA) and 1,2-dioleoyloxy-N,N - Dimethyl-3-aminopropane (DLenDMA).

Examples of polycationic lipids include tetramethyltetrapalmitoylspermine (TMTPS), tetramethyltetraoleylspermine (TMTOS), tetramethyltetralaurylspermine (TMTLS), tetramethyltetramyristine Spermine (TMTMS), Tetramethyldioleylspermine (TMDOS), their pharmaceutically acceptable salts and mixtures thereof.

Examples of polycationic lipids include 2,5-bis(3-aminopropylamino)-N-(2-(dioctadecylamino)-2-oxyethyl)pentanamide (DOGS); 2,5 - bis(3-aminopropylamino)-N-(2-(di(Z)-octadec-9-dienylamino)-2-oxoethyl)pentanamide (DOGS-9-en); 2,5-bis(3-aminopropylamino)-N-(2-(two(9Z,12Z)-octadeca-9,12-dienylamino)-2-oxyethyl)pentanamide ( DLinGS); 3-β-(N ⁴ -(N ¹ , N ⁸ -dicarbobenzoyloxyspermidine)carbamoyl)cholesterol (GL-67), (9Z, 9'Z)-2-( 2,5-bis(3-aminopropylamino)pentylamino)propane-1,3-diyl-dioctadec-9-enoate (DOSPER); 2,3-dioleyloxy-N -[2(spermidoylamino)ethyl]-N,N-dimethyl-1-propylammonium trifluoroacetate (DOSPA); their pharmaceutically acceptable salts and mixtures thereof.

Examples of cationic lipids include DS404-28 BGTC (CAS 182056-06-0), DOSPER (CAS 178532-92-8), GL-67 (179075-30-0), RPR209120 (CAS433292-13-8), DOGS (12050-77-7), DOGS (9-en, C18:1), DLinGS (C18:2) and DOTMA (104162-48-3).

阳离子脂质的实例描述于美国专利4,897,355、5,279,833、6,733,777、6,376,248、5,736,392、5,334,761、5,459,127、2005/0064595、5,208,036、5,264,618、5,279,833、5,283,185、5,753,613和5,785,992中。

In some embodiments, the composition is a mixture or complex of an RNA component and a DILA2 amino acid compound and a non-cationic lipid. In some embodiments, the composition is a mixture or complex of one or more RNA components and one or more DILA2 amino acid compounds and one or more non-cationic lipids.

Non-cationic lipids include neutral, amphoteric and anionic lipids. Thus, non-cationic amphoteric lipids may include cationic headgroups.

Examples of non-cationic lipids include 1,2-dilauroyl-tin-glycerol (DLG); 1,2-dimyristoyl-tin-glycerol (DMG); 1,2-dipalmitoyl-tin-glycerol ( DPG); 1,2-Distearoyl-tin-glycerol (DSG); 1,2-Dilauroyl-tin-glyceryl-3-phosphatidic acid (sodium salt; DLPA); 1,2-Dimyristyl Acyl-tin-glyceryl-3-phosphatidic acid (sodium salt; DMPA); 1,2-dipalmitoyl-tin-glyceryl-3-phosphatidic acid (sodium salt; DPPA); 1,2-distearoyl -Tin-glyceryl-3-phosphatidic acid (sodium salt; DSPA); 1,2-Diarachidoyl-tin-glycero-3-phosphocholine (DAPC); 1,2-Dilauroyl-tin-glycerol 1,2-dimyristoyl-tin-glyceryl-3-phosphocholine (DMPC); 1,2-dipalmitoyl-tin-glyceryl-O-ethyl 3-phosphocholine (chloride or triflate; DPePC); 1,2-dipalmitoyl-tin-glyceryl-3-phosphocholine (DPPC); 1,2-distearyl Acyl-tin-glyceryl-3-phosphocholine (DSPC); 1,2-Dilauroyl-tin-glycero-3-phosphoethanolamine (DLPE); 1,2-Dimyristoyl-tin-glyceryl -3-phosphoethanolamine (DMPE); 1,2-dipalmitoyl-tin-glyceryl-3-phosphoethanolamine (DPPE); 1,2-distearoyl-tin-glyceryl-3-phosphoethanolamine (DSPE ); 1,2-Dilauroyl-tin-glycero-3-phosphate glycerol (sodium salt; DLPG); 1,2-dimyristoyl-tin-glyceryl-3-phosphate glycerol (sodium salt; DMPG) ; 1,2-Dimyristoyl-tin-glyceryl-3-phosphate-tin-1-glycerol (ammonium salt; DMP-tin-1-G); 1,2-dipalmitoyl-tin-glyceryl- 3-Phosphoglycerol (sodium salt; DPPG); 1,2-Distearoyl-tin-glyceryl-3-phosphate glycerol (sodium salt; DSPG); 1,2-Distearoyl-tin-glyceryl- 3-Phospho-tin-1-glycerol (sodium salt; DSP-tin-1-G); 1,2-dipalmitoyl-tin-glyceryl-3-phospho-L-serine (sodium salt; DPPS); 1 - Palmitoyl-2-linoleoyl-tin-glycero-3-phosphocholine (PLinoPC); 1-palmitoyl-2-oleoyl-tin-glycero-3-phosphocholine (POPC); 1- Palmitoyl-2-oleoyl-tin-glycero-3-phosphoglycerol (sodium salt; POPG); 1-palmitoyl-2-oleoyl-tin-glycero-3-phosphoglycerol (sodium salt; POPG); 1-palmitoyl-2-oleoyl-tin-glycero-3-phosphoglycerol (ammonium salt; POPG); 1-palmitoyl-2-4o-tin-glyceryl-3-phosphocholine (P- PC); 1-stearoyl-2-sol-tin-glyceryl-3-phosphocholine (S-sol-PC); and mixtures thereof.

Examples of non-cationic lipids include polymeric compounds and polymeric lipid conjugates or polymeric lipids such as pegylated lipids containing PEG regions of molecular weight 300, 500, 1000, 1500, 2000, 3500 or 5000 , including polyethylene glycol, N-(carbonyl-methoxypolyethylene glycol-2000)-1,2-dimyristoyl-tin-glyceryl-3-phosphoethanolamine (sodium salt; DMPE-MPEG-2000 ); N-(carbonyl-methoxypolyethylene glycol-5000)-1,2-dimyristoyl-tin-glyceryl-3-phosphoethanolamine (sodium salt; DMPE-MPEG-5000); N-( Carbonyl-methoxypolyethylene glycol 2000)-1,2-dipalmitoyl-tin-glyceryl-3-phosphoethanolamine (sodium salt; DPPE-MPEG-2000); N-(carbonyl-methoxypolyethylene glycol Diol 5000)-1,2-dipalmitoyl-tin-glyceryl-3-phosphoethanolamine (sodium salt; DPPE-MPEG-5000); N-(carbonyl-methoxypolyethylene glycol 750)-1, 2-Distearoyl-tin-glyceryl-3-phosphoethanolamine (sodium salt; DSPE-MPEG-750); N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-distearoyl -Tin-glyceryl-3-phosphoethanolamine (sodium salt; DSPE-MPEG-2000); N-(carbonyl-methoxypolyethylene glycol 5000)-1,2-distearoyl-tin-glyceryl- 3-Phosphoethanolamine (sodium salt; DSPE-MPEG-5000); sodium cholesterol sulfate (SCS); their pharmaceutically acceptable salts and mixtures thereof.

Examples of non-cationic lipids include polymeric lipids such as DOPE-PEG, DLPE-PEG, DDPE-PEG DLinPE-PEG and diacylglycerol-PEG-2000 or diacylglycerol-PEG-5000.

Examples of non-cationic lipids include polymeric lipids, such as multibranched pegylated compounds, eg DSPE-PTE020 and DSPE-AM0530K.

Examples of non-cationic lipids include polymeric lipids, such as DSPE-PG8G polyglycerol lipids.

Examples of non-cationic lipids include dioleoylphosphatidylethanolamine (DOPE), diphytanoylphosphatidylethanolamine (DPhPE), 1,2-dioleoyl-tin-glycero-3-phosphocholine (DOPC) and 1,2-Diphytanoyl-tin-glycero-3-phosphocholine (DPhPC).

Examples of non-cationic lipids include cholesterol, sterols and steroids such as sterane, estrane, androstane, pregnane, cholane, cholestane, ergostane, campesterane, porosterane, stigmasterane, Corostane, lanosterane, cyclopolane, and any sterol or zoosterol derivatives of the foregoing and their biological intermediates and precursors, which include, for example, cholesterol, lanosterol, stigmasterol, di Hydrolanosterol, zymosterol, zymosterol, streptosterol, 7-dehydrocholesterol and mixtures and derivatives thereof.

Examples of non-cationic lipids include pegylated cholesterol and cholestane 3-oxo(C1-22 acyl) derivatives such as cholesterol acetate, cholesterol arachidonic acid ester, cholesterol butyrate, cholesterol hexanoate Cholesterol Caprylate, Cholesterol Caprylate, Cholesterol N-Decanoate, Cholesterol Lauryl Decanoate, Cholesterol Myristate, Cholesterol Palmitate, Cholesterol Behenate, Cholesterol Stearate, Cholesterol Neuralate, Cholesterol Nonanoate, Cholesterol n-valerate, Cholesterol oleate, Cholesterol elaidate, Cholesterol erucate, Cholesterol enanthate, Cholesterol elatate, Cholesterol linoleate and their mixtures and derivatives .

Examples of non-cationic lipids include compounds derived from phytosterols, including phytosterols, beta-sitosterol, campesterol, ergosterol, brassicasterol, delta-7-stigmasterol, delta-7-avenasterol, and mixtures thereof and derivatives.

Examples of non-cationic lipids include bile acid, cholic acid, cheicholic acid, glycocholic acid, taurocholic acid, deoxycholic acid, lithocholic acid, methyl lithocholic acid, and mixtures and derivatives thereof.

Examples of non-cationic lipids include compounds derived from steroids, including glucocorticoids, cortisol, hydrocortisone, corticosterone, ^Δ5 -pregnenolone, progesterone, deoxycorticosterone, 17-OH - Pregnenolone, 17-OH-progesterone, 11-deoxycortisol, dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione, aldosterone, 18-hydroxycorticosterone , tetrahydrocortisone, tetrahydrocortisone, cortisone, prednisone, 6α-methylprednisone, 9α-fluoro-16α-hydroxyprednisolone, 9α-fluoro-16α-methylperidone Nisolone, 9α-fluorocortisol and their mixtures and derivatives.

Examples of non-cationic lipids include compounds derived from steroids, including androgens, testosterone, dihydrotestosterone, androstenediol, androstenedione, androstenedione, 3α, 5α - androstanediols and their mixtures and derivatives.

Examples of non-cationic lipids include compounds derived from steroids, including estrogens, estriol, estrone, estradiol, and mixtures and derivatives thereof.

Examples of non-cationic lipids include compounds derived from photosterols and vitamin D compounds.

Examples of non-cationic lipids include lipids with tails ranging from C10:0 to C22:6, such as DDPE(C10:0) (CAS 253685-27-7), DLPE(C12:0) (CAS 59752-57 -7), DSPE(C18:0)(CAS 1069-79-0), DOPE(C18:1)(CAS 4004-05-1), DLinPE(C18:2)(CAS 20707-71-5), DLenPE (C18:3)(CAS 34813-40-6), DARAPE(C20:4)(CAS 5634-86-6), DDHAPE(C22:6)(CAS 123284-81-1), DPhPE(16:0[ (CH ₃ ) ₄ ]) (CAS 201036-16-0).

Examples of anionic lipids include phosphatidylserine, phosphatidic acid, phosphatidylcholine, platelet activating factor (PAF), phosphatidylethanolamine, phosphatidyl-DL-glycerol, phosphatidylinositol, phosphatidylinositol (pi(4) p, pi(4,5)p2), cardiolipin (sodium salt), lysophospholipids, hydrogenated phospholipids, sphingolipids, gangliosides, phytosphingosine, sphingosine, their pharmaceutically acceptable salts and their mixture.

Applications for the delivery of RNA therapeutics

In some aspects, the invention relates generally to the fields of regulatory RNA and RNA interference, delivery of antisense therapeutics and RNA therapeutics. More specifically, the present invention relates to compositions and formulations of ribonucleic acids, and their use in medicaments and as therapeutic agents for delivery. The present invention generally relates to methods of using ribonucleic acid in RNA interference to effect gene-specific inhibition of gene expression in cells or in mammals, thereby altering a disease state or phenotype.

RNA interference refers to a method of sequence-specific post-transcriptional gene silencing mediated by double-stranded RNA (dsRNA) called short interfering RNA (siRNA). See Fire et al., Nature 391:806, 1998, and Hamilton et al., Science 286:950-951, 1999. RNAi is shared by diverse bacterial groups and phyla and is believed to be an evolutionarily conserved cellular defense mechanism against foreign gene expression. See Fire et al., Trends Genet. 15:358,1999.

RNAi is thus a ubiquitous endogenous mechanism that utilizes small non-coding RNAs to silence gene expression. See Dykxhoorn, D.M. and J. Lieberman, Annu. Rev. Biomed. Eng. 8:377-402, 2006. RNAi can regulate important genes involved in cell death, differentiation and development. RNAi also protects the genome from genetic elements encoded by transposons and viruses. When siRNA is introduced into cells, it can bind to endogenous RNAi structures, thereby disrupting the expression of mRNAs containing highly specific complementary sequences. Any disease-causing gene and any cell type or tissue can potentially be targeted. This technology has been rapidly used in gene function analysis and drug target discovery and confirmation. Controlled RNAi also holds great therapeutic promise, although there are many obstacles to delivering siRNA into cells in vivo.

Although the mechanism of RNAi has not been fully characterized, it is thought to be through cleavage of the target mRNA. The RNAi response involves an endonuclease complex called the RNA-induced silencing complex (RISC), which mediates cleavage of single-stranded RNA complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA occurs in the middle of the region complementary to the antisense strand of the siRNA duplex (Elbashir et al., Genes Dev. 15:188, 2001).

One method of achieving RNAi is to introduce siRNA into cells or express siRNA in cells. Another approach is to utilize the endogenous RNase III enzyme called Dicer. One activity of Dicer is the processing of long dsRNA into siRNA. See Hamilton et al., Science 286:950-951, 1999; Berstein et al., Nature 409:363, 2001. Dicer-derived siRNAs are typically about 21 to 23 nucleotides in length and are double-helicated by about 19 base pairs. See Hamilton et al., supra; Elbashir et al., Genes Dev. 15:188, 2001. Essentially, long dsRNAs can be introduced into cells as precursors to siRNAs.

The present invention provides a series of compositions, formulations and methods comprising the combination of regulatory RNA, interfering nucleic acid or precursors thereof with various components including DILA2 amino acid compounds, lipids and natural or synthetic polymers combination.

As used herein, the term "dsRNA" refers to any double-stranded RNA molecule capable of inhibiting or down-regulating gene expression, for example by promoting RNA interference ("RNAi" or "iRNA") or gene silencing in a sequence-specific manner. The dsRNA of the present invention may be a substrate suitable for Dicer, or a substrate suitable for binding to RISC to mediate gene silencing through RNAi. One or both strands of the dsRNA may further include terminal phosphate groups, such as 5'-phosphate or 5',3'-diphosphate. Herein, the dsRNA molecule may further include, in addition to at least one ribonucleotide, substituted, chemically modified nucleotides and non-nucleotides.

Examples of dsRNA molecules can be found in, eg, US Patent Application No. 11/681,725, US Patent Nos. 7,022,828 and 7,034,009, and PCT International Application Publication No. WO/2003/070897.

Examples of nucleic acid reagents of the present invention may comprise one or more acyclic monomers as described in PCT International Application Publication No. WO2008/147824. Examples of the acyclic monomer include monomer D to monomer J shown in Figures 1 and 2 of WO2008/147824.

Examples of active agents of the invention include nucleic acid molecules comprising the acyclic monomers of WO2008/147824.

Examples of agents of the invention include UsiRNA. UsiRNA is an siRNA containing UNA, where UNA is an "unlocked nucleobase analog". Acyclic Monomer D to Acyclic Monomer J of WO2008/147824 are examples of UNA.

See US Patent Nos. 6,403,566, 6,509,320, 6,479,463, 6,191,266, 6,083,482, 5,712,378, and 5,681,940 for examples of modified nucleotides. Modified nucleotides can have the following structures:

Wherein, X is O or _CH2 , Y is O, and Z is _CH2 ; _R1 is selected from the following groups: adenine, cytosine, guanine, hypoxanthine, uracil, thymine and heterocycle, wherein the heterocycle is selected from the group consisting of substituted 1,3-diazine, unsubstituted 1,3-diazine and unsubstituted 7H imidazo[4,5]1,3-diazine; and R _2. R ₃ is independently selected from the group H, OH, DMTO, TBDMSO, BnO, THPO, AcO, BzO, OP(NiPr ₂ )O(CH ₂ ) ₂ CN, OPO ₃ H, diphosphate and triphosphate , wherein R ₂ and R ₃ can be PhCHO ₂ , TIPDSO ₂ or DTBSO ₂ at the same time. As used herein, the abbreviation "Ac" refers to acetyl; the abbreviation "Bn" refers to benzyl; the abbreviation "Bz" refers to benzoyl; the abbreviation "DMT" refers to dimethoxytrityl; THP" means tetrahydropyranyl; the abbreviation "TBDMS" means t-butyldimethylsilyl; the abbreviation "TIPDS" means tetraisopropyldisilyl; and the abbreviation "DTBS" means di( tert-butyl) silyl group.

In addition, as used herein, the terms "dsRNA", "RNAi inducer" and "RNAi agent" are synonymous with other terms used to describe nucleic acid molecules capable of mediating sequence-specific RNAi, including partially double-helical RNA (meroduplex RNA, mdRNA), nicked dsRNA (ndsRNA), gapped dsRNA (gdsRNA), short interfering nucleic acid (siRNA), siRNA, microRNA (miRNA), single-stranded RNA, short hairpin RNA (shRNA), short interfering oligonucleotides, short interfering substituted oligonucleotides, short interfering modified oligonucleotides, chemically modified dsRNA and post-transcriptional gene silencing RNA (ptgsRNA), and precursors of any of the above.

The term "large double-stranded (ds) RNA" refers to any double-stranded RNA greater than about 40 base pairs (bp) to about 100 bp or larger, especially up to about 300 bp to about 500 bp. Large dsRNA sequences can represent mRNA fragments or complete mRNAs. Double-stranded structures can be formed by self-complementary nucleic acid molecules or by annealing of two or more different complementary nucleic acid molecule strands.

In some aspects, a dsRNA comprises two separate oligonucleotides comprising a first strand (antisense) and a second strand (sense), wherein the antisense strand and the sense strand are self-complementary (i.e., each The strand contains a nucleotide sequence that is complementary to the nucleotide sequence of the other strand, and the two separate strands form a double helix or double-stranded structure, for example, wherein the double-stranded region is about 15 to about 24 base pairs, or about 26 to about 40 base pairs); the antisense strand includes a nucleotide sequence or portion thereof that is complementary to a nucleotide sequence in a target nucleic acid molecule (such as human mRNA); and the sense strand includes a sequence corresponding to (i.e., homologous to ) the nucleotide sequence or portion thereof in the target nucleic acid sequence (eg, corresponding to the sense strand or portion thereof of about 15 to about 25 nucleotides or about 26 to about 40 nucleotides of the target nucleic acid).

In some embodiments, a dsRNA can be assembled from a single oligonucleotide, wherein the self-complementary sense and antisense strands of the dsRNA are joined together by a nucleic acid-based linker or a non-nucleic acid-based linker. In some embodiments, the first (antisense) strand and the second (sense) strand of the dsRNA molecule are covalently linked by a nucleotide or non-nucleotide linker as described herein and known in the art. In some embodiments, a first dsRNA molecule is covalently linked to at least one second dsRNA molecule via nucleotide or non-nucleotide linkers known in the art, wherein the first dsRNA molecule can be linked to a plurality of other dsRNA molecules (identical or different) or any combination thereof. In some embodiments, the ligated dsRNA can include a third strand that forms part of a duplex with the ligated dsRNA.

In some aspects, the dsRNA molecules described herein form partially duplex RNA (mdRNA) having three or more strands, such as 'A' (first or antisense) strand, 'S1' (second) strand, and 'S2 '(third) strand, wherein the 'S1' and 'S2' strands are complementary to and form base pairs (bp) with the non-overlapping region of the 'A' strand (eg, an mdRNA may have the form A:S1S2). S1, S2 or more strands collectively contain essentially the sense strand of the 'A' strand. The double-stranded region formed by the annealing of the 'S1' and 'A' strands is distinct and non-overlapping from the double-stranded region formed by the annealing of the 'S2' and 'A' strands. The mdRNA molecule is a "gap" molecule, meaning a "gap" ranging from 0 nucleotides up to about 10 nucleotides. In some embodiments, the A:S1 duplex is separated from the A:S2 duplex by a gap arising from at least one unpaired nucleotide (up to about 10 unpaired nucleotides) of the 'A' strand, said The gap is located between the A:S1 duplex and the A:S2 duplex and is different from any one or more unpaired nucleosides at the 3'-end of one or more 'A', 'S1' or 'S2' strands acid. In some embodiments, the A:S1 duplex is separated from the A:B2 duplex by a zero-nucleotide gap between the A:S1 duplex and the A:S2 duplex (i.e., where only two A gap in which the phosphodiester bond between nucleotides is broken or deleted), which is also called a nicked dsRNA (ndsRNA). For example, A:S1S2 can consist of a dsRNA having at least two double-stranded regions totaling from about 14 base pairs to about 40 base pairs, and the double-stranded regions are separated by a gap of about 0 to about 10 nucleotides , optionally with blunt ends, or A: S1S2 can have a dsRNA composition of at least two double-stranded regions separated by a gap of up to 10 nucleotides, wherein at least one double-stranded region contains about 5 base pairs to 13 base pairs.

As described herein, dsRNAs containing three or more strands may be referred to as "partially duplex" RNAs (mdRNAs). Examples of mdRNA molecules can be found in US Provisional Patent Applications 60/934,930 and 60/973,398.

A dsRNA or large dsRNA may include substitutions or modifications, where the substitutions or modifications may be in phosphate backbone linkages, sugars, bases or nucleosides. The nucleoside substitution may include natural non-standard nucleosides (such as 5-methyluridine or 5-methylcytidine or 2-thiothymidine ribonucleoside), and the backbone, sugar or nucleoside modification Alkyl or heteroatom substitutions or additions may be included, such as methyl, alkoxyalkyl, halo, nitrogen or sulfur or other modifications known in the art.

In addition, the term "RNAi" is used herein to mean equivalent to other terms used to describe sequence-specific RNA interference, such as post-transcriptional gene silencing, translational inhibition, or epigenetics. For example, the dsRNA molecules of the invention can be used to epigenetically silence genes at the post-transcriptional level or the pre-transcriptional level or a combination thereof.

In some aspects, the invention provides compositions comprising one or more RNAi-inducing agents targeting one or more genes or target transcripts and one or more delivery components. Examples of delivery components include DILA2 amino acid compounds, lipids, peptides, polymers, polymeric lipids, and conjugates thereof.

The compositions and formulations of the invention can be used to deliver RNAi-inducing entities such as dsRNA, siRNA, mdRNA, miRNA, shRNA, or RNAi-inducing vectors into cells of intact mammalian subjects, and can be used to deliver these formulations to cells in culture.

The present invention also provides methods of delivering one or more RNAi-inducing agents or entities to cells, organs and tissues in a mammal. In some aspects, compositions comprising RNAi-inducing entities can be introduced by different routes for in vivo transport and uptake by cells within one or more organs or tissues, where expression of target transcripts can be modulated.

In general, the invention includes RNAi-inducing agents, which are useful therapeutic agents for the prevention and treatment of diseases or conditions characterized by various abnormal processes. For example, viruses that infect mammals can replicate by manipulating the cellular machinery of the host cell. See eg Fields Virology (2001). Thus, dsRNA can be used to disrupt viral pathways that control virus production or replication.

The present invention includes methods of treating or preventing viral infection in a subject by utilizing one or more therapeutic RNAi-inducing agents that have broad-spectrum potency against target viral strains. The RNAi inducing agent of the present invention can target known virus variant strains or viral gene sequences in virus variants, and exhibit sequence-specific gene silencing of target viral genes in these variants. For example, RNAi inducers can target and demonstrate efficacy against seasonal strains of influenza virus as well as against influenza variants.

The compositions and formulations of the invention can be used to deliver pharmaceutical agents or bioactive agents to various cells in vitro. Examples of cells for in vitro delivery include epidermal cells such as A549, permanent cell lines such as HeLa, hepatoma cells such as HepG2, rat gliosarcoma cells such as 9L/LacZ, human monocytes such as THP-1, Ma-Darby canine Kidney cells (MDCK), various fibroblast cell lines, and primary cells cultured in the presence or absence of various sera, etc.

The compositions and formulations of the invention can be used to deliver pharmaceutical agents or bioactive agents to various cells, tissues or organs in vivo. Methods of delivering agents in vivo include topical, enteral and parenteral routes. Examples of methods for in vivo delivery of agents include particle or droplet inhalation, nasal or nasopharyngeal drops, particle or suspension delivery, transdermal and transmucosal routes, and via intramuscular, subcutaneous, intravenous, intraarterial, intracardiac, sheath intraosseous, intraperitoneal and epidural injection or infusion routes.

In some embodiments, an agent may be administered ex vivo by direct contact with cells, tissues or organs derived from a mammalian subject.

A pharmaceutical agent or bioactive agent delivered using a composition or formulation of the invention may be in any form including, for example, pure form, crystalline form, solid form, nanoparticle, condensed form, complex form, or conjugate form.

The present invention also provides methods of delivering one or more RNAi-inducing entities to organs and tissues in a mammal. In some embodiments, a composition comprising an RNAi-inducing entity, one or more DILA2 amino acid compounds, and one or more lipid components can be introduced through different routes so that it is transported in vivo and absorbed by one or more Cellular uptake within an organ or tissue where expression of target transcripts can be regulated.

The present invention provides pharmaceutically acceptable nucleic acid compositions containing various DILA2 amino acid compounds or lipids for therapeutic delivery of nucleic acids and gene silencing RNAs. In particular, the present invention provides compositions and methods for in vitro and in vivo delivery of dsRNA to reduce, downregulate or silence translation or gene expression of a target nucleic acid sequence. These compositions and methods are useful for the prevention and/or treatment of disease in mammals. In exemplary methods of the invention, a ribonucleic acid molecule, such as siRNA or shRNA, is contacted with a DILA2 amino acid compound to formulate a composition that can be administered to a cell or a subject (eg, a mammal). In some embodiments, the invention provides methods of delivering siRNA or shRNA intracellularly by contacting a nucleic acid-containing composition with the cell.

In exemplary embodiments, the invention includes compositions comprising a nucleic acid molecule such as double-stranded RNA (dsRNA), short interfering RNA (siRNA) or short hairpin RNA (shRNA), which is combined with a DILA2 amino acid compound and Polymeric lipids are mixed or complexed to form compositions that enhance intracellular delivery of nucleic acid molecules. In some embodiments, a delivery composition of the invention may include dsRNA and one, two or more DILA2 amino acid compounds that may be cationic or non-cationic. In certain variations, a delivery composition may comprise dsRNA, a DILA2 amino acid compound, and one or more polymeric lipids. In some embodiments, a delivery composition can include dsRNA, one or more DILA2 amino acid compounds, one or more lipids, and one or more polymeric lipids. Compositions of the invention can form stable particles that can incorporate dsRNA as an interfering RNA agent. Compositions and formulations of the invention may include further delivery enhancing components or excipients.

In some embodiments, compositions of the invention comprise stable RNA-containing particles having a diameter of about 5 nm to about 400 nm. In some embodiments, the particles can have a uniform diameter from about 10 nm to about 300 nm. In some embodiments, the particles can have a uniform diameter of about 50 nm to about 150 nm.

In exemplary compositions of the invention, double-stranded RNA can be mixed or complexed with DILA2 amino acid compounds to form compositions that enhance intracellular delivery of dsRNA compared to contacting target cells with naked dsRNA.

In some embodiments, the compositions of the present invention may comprise from about 0.5% to about 70% (mol %) of one or more DILA2 amino acid compounds based on the total amount of DILA2 amino acid compound and lipid (if present), and include any A delivery enhancing component that polymerizes the component but does not include the RNA component. In some embodiments, compositions of the present invention may include from about 10% to about 55% of one or more DILA2 amino acid compounds. In some embodiments, compositions of the present invention may include from about 15% to about 35% of one or more DILA2 amino acid compounds.

In some embodiments, the compositions of the present invention may comprise from about 2% to about 95% (mole %) of one or more non-cationic lipids based on the total amount of DILA2 amino acid compound and lipid (if present), and include Any polymeric component but excluding the delivery enhancing component of the RNA component. In some embodiments, the compositions of the present invention may comprise from about 20% to about 75%, or from about 45% to about 75%, or from about 45% to about 55%, of one or more non-cationic lipids. In some embodiments, the compositions of the present invention may comprise from about 10% to about 50% of one or more non-cationic lipids.

In some embodiments, the compositions of the present invention may comprise from about 0.2% to about 20% (mol %) of one or more polymeric lipids based on the total amount of DILA2 amino acid compound and lipid (if present), and include any A delivery enhancing component that polymerizes the component but does not include the RNA component. In some embodiments, compositions of the present invention may include from about 0.5% to about 10% of one or more polymeric lipids. In some embodiments, the compositions of the present invention may comprise from about 1% to about 5% of the composition of one or more polymeric lipids.

Nucleic acid therapeutic compositions and uses

In some embodiments, the present invention provides a method of treating a disease or condition in a mammalian subject. A subject comprising an interfering RNA, a DILA2 amino acid compound, a non-cationic lipid, a polymeric lipid and Compositions of the invention in a therapeutically effective amount of one or more delivery enhancing components or excipients.

The invention includes methods of treating pulmonary disorders, such as respiratory distress, asthma, cystic fibrosis, pulmonary fibrosis, chronic obstructive pulmonary disease, bronchitis, or emphysema, by administering to a subject a therapeutically effective amount of the composition.

The present invention includes treatment including cancer, bladder cancer, liver cancer, liver disease, hypercholesterolemia, inflammatory disease, metabolic disease, inflammation, arthritis, rheumatoid arthritis, encephalitis, bone fracture, heart disease, viral disease, Methods for diseases including hepatitis and influenza.

Methods for preparing liposomes are described, for example, in G. Gregoriadis, Liposome Technology (CRCPress 1984), and M.J. Ostro, Liposomes (Marcel Dekker 1987).

The nucleic acid component, DILA2 amino acid compound and other components can first be mixed together in a suitable medium, such as a cell culture medium, and then one or more lipids or compounds can be added to the mixture. Alternatively, the DILA2 amino acid compound can be mixed first in a suitable medium, such as a cell culture medium, before adding the nucleic acid component.

In some embodiments of the invention, the dsRNA is mixed with one or more DILA2 amino acid compounds or a combination of one or more DILA2 amino acid compounds and a non-cationic lipid.

Interfering RNA agents can also be complexed or conjugated to DILA2 amino acid compounds or polymeric lipids and mixed with one or more non-cationic lipids, or a combination of one or more non-cationic and cationic lipids.

The interfering RNA agent and the DILA2 amino acid compound can be first mixed together, followed by the addition of one or more non-cationic lipids or a combination of non-cationic and cationic lipids added to a suitable medium, such as a cell culture medium. Alternatively, the DILA2 amino acid compound and lipid fraction can be mixed first, followed by the addition of the RNA reagent in a suitable medium.

In some embodiments, the invention includes micellar dispersion compositions comprising a drug or active agent mixed or complexed with a DILA2 amino acid compound and a dispersing agent to form a composition that provides intracellular delivery of the drug or active agent.

In some embodiments, the dispersion compositions of the present invention may include one or more drugs or active agents, one or more DILA2 amino acid compounds, and one or more dispersants. In certain variations, a delivery composition may include a drug or active agent, a dispersing agent, a DILA2 amino acid compound, and optionally a polymeric lipid. The dispersion compositions of the present invention can form stable particles into which drugs or active agents can be incorporated.

In some aspects, the dispersion compositions of the present invention can comprise stable nucleic acid dispersion particles having a diameter of about 5 nm to about 400 nm. In some embodiments, the particles can have a uniform diameter from about 10 nm to about 300 nm. In some embodiments, the particles can have a uniform diameter of about 50 nm to about 150 nm.

Micellar dispersions can be used to formulate and improve the bioavailability of drugs or agents, including RNAi therapeutics. Micellar dispersions can provide dispersed droplets or nanoparticles with a hydrophobic oil-like core. Dispersed nanoparticles can be suspended in a continuous aqueous phase. Dispersed Structures Utilizing the liposomal structure to deliver active agents avoids some of the inherent disadvantages and can provide delivery advantages due to the lipophilic core.

The present invention provides a series of micellar dispersion compositions comprising a DILA2 amino acid compound or lipid and a dispersing agent for use in drugs or medicaments and for delivery and administration of RNA formulations.

Examples of dispersants include synthetic compounds including polyoxyglycerides such as pegylated capryl glycerides, ethoxydiglycol, pegylated fatty acid glycerides, diethylene glycol monoethyl ether, and mixtures thereof . Examples of dispersants include LABRAFIL, LABRASOL, ARLATONE, TRANSCUTOL and mixtures thereof. Examples of dispersants include synthetic compounds such as alkylphosphate-N-methylethanolamine and alkoylsarcosine. Examples of dispersants include FOS-MEA and CRODASINIC.

In some embodiments, the delivery compositions of the present invention may include a drug or active agent, one or more oils, one or more DILA2 amino acid compounds, and emulsifiers and stabilizers lipids. In certain variations, delivery compositions may include drugs or active agents, oils, lipid emulsifiers, DILA2 amino acid compounds, non-cationic lipids, and polymeric lipids.

The compositions of the invention can form stable particles into which drugs or active agents can be incorporated. In some aspects, compositions of the invention include stable drug or active agent emulsion particles having a diameter of from about 5 nm to about 400 nm. In some embodiments, the particles have a uniform diameter from about 10 nm to about 300 nm. In some embodiments, the particles have a uniform diameter of about 50 nm to about 150 nm.

In some embodiments, a drug or active agent can be mixed or complexed with oils, emulsifiers, DILA2 amino acid compounds, and polymer-stabilized lipids to form compositions that enhance intracellular delivery of the drug or active agent.

Oil-in-water emulsions can be used to formulate and increase the bioavailability of drugs or agents, including RNAi therapeutics.

Oil-in-water emulsions can provide emulsion droplets or nanoparticles with a DILA2 amino acid compound or lipid layer surrounding a hydrophobic oil core. Emulsion droplets or nanoparticles can be suspended in a continuous aqueous phase. Utilizing the liposomal structure for the delivery of active agents, the emulsion structure can avoid some of the inherent disadvantages and offer delivery advantages due to the lipophilic core.

The present invention provides a series of novel emulsion compositions including novel composition and use of interfering RNA agents with oils, emulsifiers, DILA2 amino acid compounds and lipid components.

Examples of oils include synthetic oils, propylene glycol fatty acid esters, glycol ethers, glycerin oils, cholesterol oils, vegetable oils, peanut oils, essential oils, mineral oils, lipid-soluble compounds such as tocopherol and vitamin E, and mixtures thereof. Examples of oils include synthetic oils such as CAPRYOL 90 (propylene glycol monoester), CAPRYOL PGMC (propylene glycol monoester), LABRAFAC PC (propylene glycol monoester), LABRAFAC PG (propylene glycol diester), LAUROGLYCOL 90 (propylene glycol monoester), LAUROGLYCOLFCC (propylene glycol monoester), PLUROL OLEIQUE CC 497 (propylene glycol monoester), LABRAFACLIPOPHILE WL 1349 (triglyceride), PECEOL (monoglyceride), MAISINE 35-1 (monoglyceride) and mixtures thereof.

Compositions and methods for RNA therapeutics

The present invention provides compositions and methods for modulating gene expression using regulatory RNA, such as by RNA interference. Compositions of the invention can deliver ribonucleic acid agents to cells capable of producing an RNAi response. Examples of nucleic acid reagents that can be used in the present invention include double-stranded nucleic acids, modified or degradation-resistant nucleic acids, RNA, siRNA, siRNA, shRNA, miRNA, piRNA, RNA antagonists, single-stranded nucleic acids, DNA-RNA chimeras, reverse Sense nucleic acids and ribozymes. Herein, the terms siRNA, siRNA and shRNA include siRNA, precursors of siRNA and shRNA, respectively. For example, the term siRNA includes RNA or double-stranded RNA suitable as Dicer substrates.

The ribonucleic acid reagents useful in the present invention can target various genes. Examples of human genes suitable as targets include TNF, FLT1, VEGF family, ERBB family, PDGFR family, BCR-ABL, and MAPK family, among others. Examples of human genes suitable as targets and their nucleic acid sequences include those described in: PCT/US08/55333, PCT/US08/55339, PCT/US08/55340, PCT/US08/55341, PCT/US08/ 55350, PCT/US08/55353, PCT/US08/55356, PCT/US08/55357, PCT/US08/55360, PCT/US08/55362, PCT/US08/55365, PCT/US08/55366, PCT/US08/55369, PCT/US08/55370, PCT/US08/55371, PCT/US08/55372, PCT/US08/55373, PCT/US08/55374, PCT/US08/55375, PCT/US08/55376, PCT/US08/55377, PCT/ US08/55378, PCT/US08/55380, PCT/US08/55381, PCT/US08/55382, PCT/US08/55383, PCT/US08/55385, PCT/US08/55386, PCT/US08/55505, PCT/US08/ 55511, PCT/US08/55515, PCT/US08/55516, PCT/US08/55519, PCT/US08/55524, PCT/US08/55526, PCT/US08/55527, PCT/US08/55532, PCT/US08/55533, PCT/US08/55542, PCT/US08/55548, PCT/US08/55550, PCT/US08/55551, PCT/US08/55554, PCT/US08/55556, PCT/US08/55560, PCT/US08/55563, PCT/ US08/55597, PCT/US08/55599, PCT/US08/55601, PCT/US08/55603, PCT/US08/55604, PCT/US08/55606, PCT/US08/55608, PCT/US08/55611, PCT/US08/ 55612, PCT/US08/55615, PCT/US08/55618, PCT/US08/55622, PCT/US08/55625, PCT/US08/55627, PCT/US08/55631, PCT/US08/55635, PCT/US08/55644, PCT/US08/55649, PCT/US0 8/55651, PCT/US08/55662, PCT/US08/55672, PCT/US08/55676, PCT/US08/55678, PCT/US08/55695, PCT/US08/55697, PCT/US08/55698, PCT/US08/ 55701, PCT/US08/55704, PCT/US08/55708, PCT/US08/55709, and PCT/US08/55711.

The RNA of the present invention to be delivered may have a sequence complementary to a viral gene region. For example, certain compositions and methods of the invention can be used to modulate expression of the viral genome of influenza virus. In some embodiments, the present invention provides compositions and methods for modulating the expression and infectious activity of influenza virus by RNA interference. Expression and/or activity of influenza virus can be modulated by delivery to cells of short interfering RNA molecules, eg, having a sequence complementary to the RNA polymerase subunit region of influenza virus. For examples of RNA targeting influenza virus, see US Patent Publication No. 20070213293 A1.

In some embodiments, the present invention provides combinations that inhibit expression of target transcripts in a subject by administering to the subject an effective amount of an RNAi-inducing compound, such as a short interfering oligonucleotide molecule or precursor thereof. things and methods. RNAi utilizes small interfering RNA (siRNA) to target messenger RNA (mRNA) and impair translation. The siRNA used in the present invention may be a Dicer-processed precursor, such as a long dsRNA processed into siRNA. The present invention provides methods of treating or preventing diseases or disorders associated with the expression of target transcripts or the activity of peptides or proteins encoded by target transcripts.

RNAi-based therapeutic strategies can treat a variety of diseases by shutting down the growth or function of viruses or microorganisms, as well as by shutting down the function of endogenous gene products in disease pathways.

In some embodiments, the present invention provides novel compositions and methods for the delivery of RNAi-inducing entities such as short interfering oligonucleotide molecules and precursors thereof. In particular, the invention provides compositions comprising RNAi-inducing entities targeting one or more transcripts to cells, tissues and/or organs of a subject.

siRNA can be two RNA strands with a region of complementarity about 19 nucleotides in length. The siRNA optionally includes one or two single-stranded overhangs or loops.

shRNA can be a single RNA strand with self-complementary regions. A single RNA strand can form a hairpin structure with a stem and loop and optionally one or more non-pairing portions at the 5' and/or 3' portion of the RNA.

The active therapeutic agent can be a chemically modified RNA with improved resistance to in vivo nuclease degradation and/or improved cellular uptake that retains RNAi activity.

The siRNA agent of the present invention may have a sequence complementary to a target gene region. The siRNA of the present invention may have a length of 29 to 50 base pairs, such as a dsRNA having a sequence complementary to a target gene region. Alternatively, the double-stranded nucleic acid can be dsDNA.

In some embodiments, the active agent can be a short interfering nucleic acid (siRNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA, or short hairpin RNA (shRNA) capable of modulating expression of a gene product.

Comparing methods and compositions are provided that target the expression of one or more different genes associated with a particular disease condition in a subject, including genes known to be aberrantly expressed due to determinants or contributing factors associated with the selected disease condition Any of the multiple genes added.

The RNAi-inducing compounds of the present invention can be used in combination with other known therapies to treat diseases.

In some embodiments, the invention relates to combinations comprising a small nucleic acid molecule (such as short interfering nucleic acid, short interfering RNA, double-stranded RNA, microRNA, or short hairpin RNA) mixed or complexed or conjugated with a delivery-enhancing compound things.

As used herein, the terms "regulatory RNA", "short interfering nucleic acid", "siRNA", "short interfering RNA", "short interfering oligonucleotide molecule" and "chemically modified short interfering nucleic acid molecule" Refers to any nucleic acid molecule that modulates, inhibits or downregulates gene expression or, for example, viral replication by mediating RNA interference (RNAi) or gene silencing in a sequence-specific manner. Regulatory RNAs include antagonists of single-stranded RNA.

In some embodiments, siRNA is a double-stranded polynucleotide molecule containing self-complementary sense and antisense regions, wherein the antisense region contains nucleosides complementary to the nucleotide sequence in the target ribonucleic acid molecule for down-regulation of expression acid sequence or part thereof, and the sense region comprises a nucleotide sequence corresponding to (ie, it is substantially identical in sequence) the target ribonucleic acid sequence or part thereof.

Herein, "siRNA" refers to small interfering ribonucleic acid, ie a relatively short length of double-stranded nucleic acid or optionally a longer precursor thereof. In some embodiments, the length of siRNA useful in the present invention is preferably about 20 to 50 bp in length. However, there is no particular limitation on the length of usable siRNA (including siRNA). For example, the siRNA may initially exist in the cell in a precursor form that is substantially different from the final or processed form of the siRNA that exhibits and exerts gene silencing activity when or after delivery to the target cell. For example, a precursor form of an siRNA can include precursor sequence elements that are processed, degraded, altered, or cleaved upon or after delivery to produce an siRNA that is active in the cell to mediate gene silencing. In some embodiments, useful siRNAs have precursors of, for example, about 100 to 200 base pairs, or 50 to 100 base pairs, or less than about 50 base pairs in length, which produce useful Active, processed siRNA. In other embodiments, useful siRNAs or pre-siRNAs are about 10 to 49 bp or 15 to 35 bp or about 21 to 30 bp in length.

In some embodiments of the invention, polynucleotide delivery enhancing polypeptides may be used to facilitate delivery of large nucleic acid precursors of nucleic acid molecules, including siRNA. For example, the methods and compositions herein can be used to enhance the delivery of larger nucleic acids that represent "precursors" to desired siRNAs, wherein precursor amino acids can be cleaved or processed before, during, or after delivery to target cells to form Active siRNAs that regulate gene expression in target cells.

For example, the dsRNA precursor polynucleotide of choice may be a circular single-stranded polynucleotide having two or more loop structures and a stem containing self-complementary sense and antisense regions, wherein the antisense region contains A nucleotide sequence or part thereof complementary to the nucleotide sequence in the target nucleic acid molecule, and the sense region has a nucleotide sequence corresponding to the target nucleic acid sequence or part thereof, and wherein the circular polynucleotide can be are processed in vivo or in vitro to produce active dsRNA molecules capable of inducing RNAi.

siRNA molecules of the invention, particularly in non-precursor form, may be less than 30 base pairs or about 17 to 19 bp or 19 to 21 bp or 21 to 23 bp.

siRNA can mediate selective gene silencing in mammalian systems. Hairpin RNAs with short loops and 19 to 27 base pairs within the stem also selectively silence the expression of genes homologous to sequences within the double-stranded stem. Mammalian cells can convert short hairpin RNAs to siRNAs to mediate selective gene silencing.

RISC mediates cleavage of single-stranded RNA with a sequence complementary to the antisense strand of the siRNA duplex. Cleavage of the target RNA occurs in a region complementary to the antisense strand of the siRNA duplex. 21 nucleotide siRNA duplexes are generally most active when they contain a two nucleotide 3'-overhang.

Replacing the 3′-overhang segment of a 21-mer siRNA duplex with a 2-nucleotide 3′ overhang with a deoxyribonucleotide may have no adverse effect on RNAi activity. Replacing up to 4 nucleotides at each end of the siRNA with deoxyribonucleotides is acceptable.

Alternatively, siRNAs may be delivered as single or multiple transcripts expressed by polynucleotide vectors encoding the single or multiple siRNAs and directed for their expression in target cells. In these embodiments, the double-stranded portion of the siRNA final transcript to be expressed in the target cell can be, for example, 15 to 49 bp, 15 to 35 bp, or about 21 to 30 bp long.

In some embodiments of the invention, the double-stranded region of the siRNA in which the two strands pair can include a bulge or a mismatched portion, or a bulge and a mismatched portion. The double-stranded portion of an siRNA in which the two strands are paired is not limited to perfectly paired stretches of nucleotides, and may contain fragments due to, for example, mismatches (the corresponding nucleotides are not complementary), bulges (the corresponding complementary core is missing on one strand) nucleotides) or unpaired portions caused by overhangs. Unpaired portions can be included without interfering with siRNA formation. In some embodiments, a "knob" can comprise 1 to 2 unpaired nucleotides, and the double-stranded region of an siRNA wherein the two strands are paired can comprise about 1 to 7 or about 1 to 5 bulges. Additionally, "mismatched" portions included within the double-stranded region of the siRNA may be present in an amount of about 1 to 7 or about 1 to 5. The most common case of a mismatch is when one nucleotide is guanine and the other is uracil. The mismatches may be due to mutations of C to T, G to A, or a mixture thereof, as in the corresponding DNA encoding the sense RNA, but other causes are also contemplated.

The terminal structure of the siRNA of the present invention may be a blunt end or a cohesive (overhanging) end as long as the siRNA maintains its activity of silencing target gene expression. Sticky (overhang) end structures are not limited to 3' overhangs, and include 5' overhang structures as long as they retain the activity of inducing gene silencing. In addition, the number of overhanging nucleotides is not limited to 2 or 3 nucleotides, but may be any number of nucleotides as long as it maintains the activity of inducing gene silencing. For example, an overhang can comprise 1 to about 8 nucleotides or 2 to 4 nucleotides.

The length of siRNAs with overhang structures can be expressed as the paired duplex portion and any overhang at each end. For example, a 25/27-mer siRNA duplex with a 2-bp 3' antisense overhang has a 25-mer sense strand and a 27-mer antisense strand, where the paired portion is 25 bp in length.

Any overhang sequence may have low specificity for the target gene and may not be complementary (antisense) or identical (sense) to the target gene sequence. As long as the siRNA retains gene silencing activity, it may include low molecular weight structures such as natural RNA molecules (eg tRNA, rRNA, viral RNA), or artificial RNA molecules in the overhang portion.

The terminal structure of siRNA may have a stem-loop structure in which one terminal of a double-stranded nucleic acid is connected by a linker nucleic acid such as a linker RNA. The length of the double stranded region (stem portion) may be eg 15 to 49 bp or 15 to 35 bp or about 21 to 30 bp. Alternatively, the length of the double-stranded region that is the final transcription product of the siRNA expressed in the target cell may be, for example, about 15 to 49 bp or 15 to 35 bp or about 21 to 30 bp.

siRNA may comprise a single-stranded polynucleotide or portion thereof having a nucleotide sequence complementary to a nucleotide sequence in a target nucleic acid molecule, wherein the single-stranded polynucleotide may comprise a terminal phosphate group such as a 5'-phosphate (see e.g. Martinez et al., Cell. 110:563-574, 2002, and Schwarz et al., Molecular Cell 10:537-568, 2002), or 5',3'-diphosphate.

Herein, the term siRNA is not limited to molecules containing only naturally occurring RNA or DNA, but also includes chemically modified nucleotides and non-nucleotides. In some embodiments, short interfering nucleic acid molecules of the invention lack 2'-hydroxyl (2'-OH) containing nucleotides. In some embodiments, the short interfering nucleic acid does not require the presence of nucleotides with a 2'-hydroxyl group for mediating RNAi, etc., and as such, the short interfering nucleic acid molecules of the invention optionally do not include any ribonucleosides Acids (such as nucleotides with a 2'-OH group). However, siRNA molecules that do not require the presence of ribonucleotides in the siRNA molecule to maintain RNAi may have one or more attached linkers or other attached or bound groups, entities or contain a 2'-OH group. A chain of one or more nucleotides. The siRNA molecule can comprise ribonucleotides at at least about 5%, 10%, 20%, 30%, 40%, or 50% of the nucleotide positions.

As used herein, the term siRNA includes nucleic acid molecules capable of mediating sequence-specific RNAi, for example, short interfering RNA (siRNA) molecules, double-stranded RNA (dsRNA) molecules, microRNA molecules, short hairpin RNA (shRNA) molecules, Short interfering oligonucleotide molecules, short interfering nucleic acid molecules, short interfering modified oligonucleotide molecules, chemically modified siRNA molecules and post-transcriptional gene silencing RNA (ptgsRNA) molecules, etc.

In some embodiments, siRNA molecules comprise separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linker molecules, or by ionic interactions, hydrogen bonds, van der Waals Interactions, hydrophobic interactions and/or stacking interactions are non-covalently linked.

An "antisense RNA" is an RNA strand having a sequence complementary to a target gene mRNA, which is capable of inducing RNAi by binding to the target gene mRNA.

A "sense RNA" is an RNA strand that has a sequence that is complementary to an antisense RNA and that anneals to its complementary antisense RNA to form siRNA.

Herein, the term "RNAi construct" or "RNAi precursor" refers to RNAi-inducing compounds such as small interfering RNA (siRNA), hairpin RNA and other RNA species that can be cleaved in vivo to form siRNA. RNAi precursors herein also include expression vectors (also referred to as RNAi expression vectors) that produce transcripts that form dsRNA or hairpin RNA in cells, and/or that produce transcripts that produce siRNA in vivo.

Short interfering hybrid (siHybrid) molecules are double-stranded nucleic acids with similar functions to siRNA. Substitutions are double-stranded RNA molecules, and short interfering hybrids consist of RNA strands and DNA strands. Preferably, the RNA strand is the antisense strand that binds the target mRNA. Short interfering hybrids generated by hybridizing DNA and RNA strands have hybridized complements, and preferably at least one 3' overhang.

siRNAs used in the present invention can be assembled from two separate oligonucleotides, one strand being the sense strand and the other being the antisense strand, wherein the antisense and sense strands are self-complementary (i.e., each strand contains The nucleotide sequence of the nucleotide sequence in the other strand; eg, where the antisense and sense strands form a double helix or double-stranded structure, eg, where the double-stranded region is about 19 base pairs). The antisense strand may include a nucleotide sequence or a portion thereof that is complementary to a nucleotide sequence in a target nucleic acid molecule, and the sense strand may include a nucleotide sequence or a portion thereof that corresponds to the target nucleic acid sequence. Alternatively, siRNAs can be assembled from a single oligonucleotide, wherein the self-complementary sense and antisense regions of the siRNA are linked by a nucleic acid-based or non-nucleic acid-based linker.

In some embodiments, the siRNA for intracellular delivery can be a polynucleotide having a double helix, asymmetric duplex, hairpin, or asymmetric hairpin secondary structure with a self-complementary sense region and an antisense region, wherein the antisense region contains a nucleotide sequence or part thereof that is complementary to the nucleotide sequence in the isolated target nucleic acid molecule, and the sense region contains a nucleotide sequence or part thereof corresponding to the target nucleic acid sequence .

Examples of chemical modifications that can be made in siRNA include the introduction of phosphorothioate internucleotide linkages, 2'-deoxyribonucleotides, 2'-O-methyl ribonucleotides, 2'-deoxy-2' - Fluorinated ribonucleotides, "universal base" nucleotides, "acyclic" nucleotides, 5-C-methyl nucleotides and terminal glyceryl and/or inverted deoxyabasic residues.

The antisense region of the siRNA molecule may include a phosphorothioate internucleotide linkage on the 3'-end of the antisense region. The antisense region may comprise about 1 to about 5 phosphorothioate internucleotide linkages on the 5'-end of the antisense region. The 3'-terminal nucleotide overhang of the siRNA molecule may include ribonucleotides or deoxyribonucleotides that may be chemically modified on the nucleic acid sugar, base, or backbone. The 3'-terminal nucleotide overhang may include one or more universal base ribonucleotides. The 3'-terminal nucleotide overhang may include one or more acyclic nucleotides.

For example, chemically modified siRNAs can have 1, 2, 3, 4, 5, 6, 7, 8, or more phosphorothioate internucleotide linkages within a strand, or can have 1 to 8 or more phosphorothioate internucleotide linkages. Phosphorothioate internucleotide linkages can be present on one or both oligonucleotide strands of the siRNA duplex, eg, on the sense strand, the antisense strand, or both strands.

siRNA molecules may include one or more phosphorothioate internucleotide linkages at the 3'-end, 5'-end, or both 3'- and 5'-ends of the sense strand, the antisense strand, or both strands. For example, exemplary siRNA molecules can include 1, 2, 3, 4, 5 or more consecutive phosphorothioate internucleotide linkages at the 5'-terminus on the sense strand, the antisense strand, or both strands .

In some embodiments, the siRNA molecule includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more pyrimidine phosphorothioates on the sense strand, the antisense strand, or both strands Nucleotide bond.

In some embodiments, the siRNA molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more purine phosphorothioates on the sense strand, the antisense strand, or both strands Nucleotide bond.

siRNA molecules can include circular nucleic acid molecules, wherein the siRNA is about 38 to about 70 nucleotides in length, such as about 38, 40, 45, 50, 55, 60, 65 or 70 nucleotides, with about 18 to about 23 base pairs, such as about 18, 19, 20, 21, 22 or 23 base pairs, wherein the circular oligonucleotide forms a dumbbell-shaped structure having about 19 base pairs and 2 loops.

Circular siRNA molecules can include 2 loop motifs, wherein one or both loop portions of the siRNA molecule are biodegradable. For example, the loop portion of a circular siRNA molecule can be converted in vivo to generate a double-stranded siRNA molecule having a 3'-terminal overhang, such as a 3'-terminal nucleotide overhang comprising about 2 nucleotides.

The modified nucleotides in the siRNA molecule can be on the antisense strand, the sense strand, or both. For example, the modified nucleotide may have a Northern configuration (eg, a Northern pseudo-gyro; see, eg, Saenger, Principles of Nucleic Acid Structure, Springer-Verlag ed., 1984). Examples of nucleotides with the Northern configuration include locked nucleic acid (LNA) nucleotides (such as 2'-O, 4'-C-methylene-(D-ribofuranosyl) nucleotides), 2'- Methoxyethoxy (MOE) nucleotides, 2'-methyl-thio-ethyl, 2'-deoxy-2'-fluoronucleotides, 2'-deoxy-2'-chloronucleosides nucleotides, 2'-azido nucleotides and 2'-O-methyl nucleotides.

Chemically modified nucleotides can be made resistant to nuclease degradation while retaining the ability to mediate RNAi.

The sense strand of a double-stranded siRNA molecule can have an endcap moiety, such as an inverted deoxyabasic moiety, on the 3'-end, 5'-end, or both the 3'- and 5'-ends of the sense strand.

Examples of conjugates include the conjugates and ligands described in US Application Serial No. 10/427,160 to Vargeese et al., filed April 30, 2003, which is incorporated herein by reference in its entirety, including the figures.

In some embodiments of the invention, the conjugates can be covalently linked to chemically modified siRNA molecules via biodegradable linkers. For example, the conjugated molecule can be linked to the 3'-end of the sense strand, the antisense strand, or both strands of the chemically modified siRNA molecule.

In some embodiments, the conjugated molecule is attached to the 5'-end of the sense strand, the antisense strand, or both strands of the chemically modified siRNA molecule. In some embodiments, the conjugated molecule is linked to the 3'-end and 5'-end of the sense strand, the antisense strand, or both strands of the chemically modified siRNA molecule, or any combination thereof.

In some embodiments, conjugated molecules include molecules that facilitate delivery of chemically modified siRNA molecules into biological systems, such as cells.

In some embodiments, the conjugate molecule to which the chemically modified siRNA molecule is attached is polyethylene glycol, human serum albumin, or a ligand for a cellular receptor that mediates cellular uptake. Examples of specific conjugated molecules contemplated by the present invention capable of linking chemically modified siRNA molecules are described in Vargeese et al., US Patent Publication Nos. 20030130186 and 20040110296.

The siRNA may comprise a nucleotide, non-nucleotide or mixed nucleotide/non-nucleotide linker linking the sense region of the siRNA and the antisense region of the siRNA. In some embodiments, a nucleotide linker can be 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides in length. In some embodiments, a nucleotide linker can be a nucleic acid aptamer. As used herein, the term "adapter" or "nucleic acid aptamer" includes a nucleic acid molecule that specifically binds a target molecule, wherein the nucleic acid molecule comprises a sequence recognized by the target molecule in its natural state. Additionally, an aptamer can be a nucleic acid molecule that binds a target molecule that does not naturally bind nucleic acid.

For example, aptamers can be used to bind the ligand-binding domain of a protein, thereby preventing naturally occurring ligands from interacting with the protein. See, eg, Gold et al., Annu.Rev.Biochem.64:763, 1995; Brody and Gold, J.Biotechnol.74:5, 2000; Sun, Curr.Opin.Mol.Ther.2:100, 2000; Kusser, J. . Biotechnol. 74: 27, 2000; Hermann and Patel, Science 287: 820, 2000; and Jayasena, Clinical Chemistry 45: 1628, 1999.

Non-nucleotide linkers can be abasic nucleotides, polyethers, polyamines, polyamides, peptides, carbohydrates, lipids, polyhydrocarbons, or other polymeric compounds (e.g., such as those with 2 to 100 ethylene glycol unit of polyethylene glycol). Specific examples include those described in Seela and Kaiser, Nucleic Acids Res. 18:6353, 1990 and Nucleic Acids Res. 15:3113, 1987; Cload and Schepartz, J.Am.Chem.Soc.113: 6324, 1991; Richardson and Schepartz, J.Am.Chem.Soc. 113:5109, 1991; Ma et al., Nucleic Acids Res. 21:2585, 1993 and Biochemistry 32:1751, 1993; Durand et al., Nucleic Acids Res. 18 : 6353,1990; McCurdy et al, Nucleosides & Nucleotides 10:287,1991; Jaschke et al, Tetrahedron Lett.34:301-304,1993; Ono et al, Biochemistry 30:9914,1991; Arnold et al, International Publication No. WO89/02439; Usman et al., International Publication No. WO 95/06731; Dudycz et al., International Publication No. WO 95/11910; and Ferentz and Verdine, J. Am. Chem. Soc. 113:4000, 1991.

"Non-nucleotide linker" refers to a group or compound that can be introduced into a nucleic acid chain to replace one or more nucleotide units (including sugar and/or phosphate substitutions) and allow the remaining bases to exhibit their enzymatic activity. The group or compound may be abasic in that it does not contain a conventionally recognized nucleotide base such as adenosine, guanine, cytosine, uracil or thymine in the C1 position of a sugar.

In some embodiments, the modified siRNA molecule can have a phosphate backbone modification including one or more of phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester, morpholino, amidated amino Formate, carboxymethyl, acetamide, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal and/or alkylsilyl substitution. Examples of oligonucleotide backbone modifications are in Hunziker and Leumann, Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, pp. 331-417, 1995 and Mesmaeker et al., Novel Backbone Replacements for Oligonucleotides, in Carbohydrate Modifications in Research, A Given in ACS, pp. 24-39, 1994.

siRNA molecules that can be chemically modified can be synthesized by (a) synthesizing the two complementary strands of the siRNA molecule; and (b) annealing the two complementary strands together under conditions suitable to obtain a double-stranded siRNA molecule. In some embodiments, the synthesis of the complementary portion of the siRNA molecule is achieved by solid phase oligonucleotide synthesis, or by solid phase tandem oligonucleotide synthesis.

Oligonucleotides (such as certain modified oligonucleotides or oligonucleotide portions lacking ribonucleotides) are synthesized using methods well known in the art, such as described in: Caruthers et al., Methods in Enzymology 211:3-19, 1992; Thompson et al., International PCT Publication No. WO99/54459; Wincott et al., Nucleic Acids Res.23:2677-2684, 1995; Wincott et al., Methods Mol.Bio.74:59, 1997; Brennan et al., Biotechnol Bioeng. 61:33-45, 1998; and Brennan, US Patent No. 6,001,311. Synthesis of RNA, including certain siRNA molecules of the invention, is performed according to methods described in, for example, Usman et al., J.Am.Chem.Soc. 109:7845, 1987; Scaringe et al., Nucleic Acids Res. 18: 5433, 1990; and Wincott et al., Nucleic Acids Res. 23:2677-2684, 1995; Wincott et al., Methods Mol. Bio.74:59, 1997.

As used herein, "asymmetric hairpins" are linear siRNA molecules that contain an antisense region, a loop portion that may comprise nucleotides or non-nucleotides, and a sense region that is sufficiently compatible with The antisense region contains fewer nucleotides than the antisense region under the premise of base-pairing complementary nucleotides and forming a double helix with a loop.

As used herein, an "asymmetric duplex" is an siRNA molecule having two separate strands, containing a sense region and an antisense region, wherein the sense region has sufficient complementary nucleotides to base-pair with the antisense region. And on the premise of forming a double helix with a loop, it contains less nucleotides than the antisense region.

Herein, "regulating gene expression" refers to upregulating or downregulating the expression of a target gene, which may include upregulating or downregulating mRNA levels present in a cell or mRNA translation or synthesis of proteins or protein subunits encoded by the target gene.

The terms "inhibit", "down-regulate" or "reduce expression" shall mean herein the expression of a gene, or the level of expression of an RNA molecule encoding one or more proteins or protein subunits, or equivalent RNA molecules, or by The level or activity of one or more proteins or protein subunits encoded by the target gene is reduced below the level observed in the absence of a nucleic acid molecule (eg, siRNA) of the invention.

Herein, "gene silencing" refers to partial or complete suppression of gene expression in cells, and may also be referred to as "gene knockdown". The extent of gene silencing can be determined by methods known in the art, some of which are summarized in International Publication No. WO99/32619.

Herein, the terms "ribonucleic acid" and "RNA" refer to molecules containing at least one ribonucleotide residue. Ribonucleotides are nucleotides that have a hydroxyl group at the 2' position of the β-D-ribose-furanose moiety. These terms include double-stranded RNA, single-stranded RNA, isolated RNA, such as partially purified RNA, substantially pure RNA, synthetic RNA, recombinantly produced RNA, and RNA obtained by adding, deleting, substituting, modifying and/or changing one or Modified and altered RNA that differs from naturally occurring RNA by a number of nucleotides. Alteration of the RNA can include, for example, the addition of non-nucleotide material to, eg, the end of the siRNA, or, eg, within one or more nucleotides of the RNA.

Nucleotides in RNA molecules include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs can be referred to as analogs.

"Highly conserved sequence region" means that the nucleotide sequence of one or more regions in the target gene does not change significantly from one generation to another or from one organism to another.

"Sense region" refers to the nucleotide sequence of an siRNA molecule that has an antisense region that is complementary to the siRNA molecule. In addition, the sense region of the siRNA molecule may contain a nucleic acid sequence having homology to the target nucleic acid sequence.

"Antisense region" refers to a nucleotide sequence of an siRNA molecule that is complementary to a target nucleic acid sequence. In addition, the antisense region of the siRNA molecule can include a nucleic acid sequence that is complementary to the sense region of the siRNA molecule.

"Target nucleic acid" refers to any nucleic acid sequence whose expression or activity is to be modulated. The target nucleic acid can be DNA or RNA.

"Complementary"means that a nucleic acid can form hydrogen bonds with another nucleic acid sequence by conventional Watson-Crick or by other unconventional binding modes.

As used herein, the term "biodegradable linker" refers to a linker designed as a biodegradable linker to link one molecule to another (e.g., a biologically active molecule to an siRNA molecule or to the sense and antisense strands of an siRNA molecule). ) nucleic acid or non-nucleic acid linker molecule. Biodegradable linkers are designed such that their stability can be tuned for specific purposes, such as delivery to specific tissues or cell types. The stability of nucleic acid-based biodegradable linker molecules can be modulated in different ways, for example, by combinations of ribonucleotides, deoxyribonucleotides and chemically modified nucleotides, which Such as 2'-O-methyl, 2'-fluoro, 2'-amino, 2'-O-amino, 2'-C-allyl, 2'-O-allyl and other 2'- Modified or base-modified nucleotides. The biodegradable nucleic acid linker molecule can be a dimer, trimer, tetramer or longer nucleic acid molecule, e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, Oligonucleotides of 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides, or may include single nucleotides with phosphorus-based linkages such as phosphoramidate or phosphodiester linkages . Biodegradable nucleic acid linker molecules may also include nucleic acid backbone, nucleic acid sugar, or nucleic acid base modifications.

For 2'-modified nucleotides as described herein, "amino" refers to 2'- _NH2 or 2'-O- _NH2 , which may be modified or unmodified. Such modified groups are described, for example, in Eckstein et al., US Patent No. 5,672,695 and Matulic-Adamic et al., US Patent No. 6,248,878.

Auxiliary or complementary methods of delivering nucleic acid molecules for use in the present invention are described, for example, in Akhtar et al., Trends Cell Bio. 2:139, 1992; Biol. 16: 129-140, 1999; Hofland and Huang, Handb. Exp. Pharmacol. 137: 165 192, 1999; and Lee et al., ACS Symp. Ser. 752: 184-192, 2000. General methods for delivering enzyme nucleic acid molecules are further described in Sullivan et al., International PCT Publication No. WO 94/02595.

Nucleic acid molecules can be administered in formulations that include one or more components such as pharmaceutically acceptable carriers, diluents, excipients, adjuvants, emulsifiers, buffers, stabilizers or preservatives.

As used herein, the term "carrier" refers to a pharmaceutically acceptable solid or liquid diluent, solvent, filler or encapsulating material. Examples of carriers include saline, biological and pharmaceutical buffer systems and biologically acceptable media. Aqueous liquid carriers can include pharmaceutically acceptable additives such as acidifying agents, alkalizing agents, antimicrobial preservatives, antioxidants, buffers, chelating agents, complexing agents, solubilizers, wetting agents, solvents, suspending agents, and and/or viscosity enhancers, tonicity agents, wetting agents or other biocompatible materials. Examples of components of the above categories can be found in U.S. Pharmacopeia National Formulary, 1990, pp. 1857-1859, and Raymond C. Rowe et al., Handbook of Pharmaceutical Excipients, 5th ed., 2006, and "Remington: The Science and Practice of Pharmacy," pp. 21 Edition, 2006, edited by David B. Troy.

Examples of preservatives include phenol, methylparaben, parabens, m-cresol, thimerosal, benzalkonium chloride, and mixtures thereof.

Examples of surfactants include oleic acid, sorbitan trioleate, polysorbates, lecithins, phosphatidylcholines, various long chain diglycerides and phospholipids, and mixtures thereof.

Examples of phospholipids include phosphatidylcholine, lecithin, phosphatidylglycerol, phosphatidylinositol, phosphatidylserine, and phosphatidylethanolamine, and mixtures thereof.

Examples of dispersants include ethylenediaminetetraacetic acid.

Examples of gases include nitrogen, helium, chlorofluorocarbons (CFCs), hydrofluorocarbons (HFCs), carbon dioxide, air, and mixtures thereof.

In some embodiments, siRNA and/or polypeptides can be encapsulated into liposomes or reside inside or outside liposomes or present in liposome layers or administered by iontophoresis or added to other carriers, such as hydrogels, Cyclodextrins, biodegradable nanocapsules, bioadhesive microspheres or protein carriers. See, eg, O'Hare and Normand, PCT International Publication No. WO 00/53722. Alternatively, nucleic acid compositions can be delivered locally by direct injection or by use of an infusion pump. Direct injection of nucleic acid molecules of the present invention, whether subcutaneous, intramuscular or intradermal, can utilize standard needle and syringe techniques, or use needle-free techniques, such as Conry et al., Clin. Cancer Res. 5: 2330- 2337, 1999, and Barry et al., International PCT Publication No. WO 99/31262.

The composition of the present invention can be effectively used as a pharmaceutical preparation. A pharmaceutical agent prevents, modulates the occurrence or severity of, or treats (reduces one or more symptoms to a detectable or measurable extent) a disease state or other adverse condition in a patient.

In some embodiments, the invention provides pharmaceutical compositions and methods characterized by the presence or administration of one or more polynucleic acids, typically a polynucleic acid bound, complexed or conjugated to a DILA2 amino acid compound or lipid. One or more siRNAs can be further compatible with pharmaceutically acceptable carriers such as diluents, stabilizers or buffers.

Typically, siRNA will target a gene expressed at high levels as a determinant or contributory factor associated with a disease or adverse condition in a subject. In this case, the siRNA effectively down-regulates the expression level of the gene, thereby preventing, alleviating or reducing the severity or recurrence of one or more associated disease symptoms. Alternatively, for various disease models, the expression of the target gene may not necessarily increase with the outcome or outcome of the disease or other adverse condition, by reducing gene expression (i.e., reducing the level of selected mRNA and/or or the level of the protein product of the target gene), downregulation of the target gene will still result in a therapeutic outcome. Alternatively, an siRNA of the invention may target a gene that is less expressed, which may result in upregulation of a "downstream" gene whose expression is negatively regulated by the product or activity of the target gene.

The siRNAs of the invention may be administered in any form, such as transdermally or by local injection (e.g., locally on the site of a psoriatic plaque to treat psoriasis, or locally into the joints of patients with psoriatic arthritis or RA). Inside). In a more specific embodiment, the present invention provides methods of formulating and administering a therapeutically effective amount of siRNA directed against TNF-alpha mRNA, which siRNA effectively downregulates TNF-alpha RNA, and thereby reduces or prevents one or more TNF-alpha - Alpha-related inflammatory disorders. Comparative methods and compositions are provided that target the expression of one or more different genes associated with selected disease states in animal subjects, including those whose expression is known to vary with determinants or contributors associated with selected disease states. Any of a large number of genes for which a factor increases abnormally.

The compositions of the present invention can also be formulated and used as tablets, capsules or elixirs for oral administration, suppositories for rectal administration, sterile solutions, suspensions for injectable administration, and other forms known in the art.

A pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, eg, systemic administration to a cell or a patient, including eg a human. The suitable form depends in part on the use or route of entry, eg oral, transdermal, epidermal or by injection. The form does not prevent the composition or formulation from reaching the target cell (ie, the cell to which delivery of the negatively charged nucleic acid is desired). For example, a pharmacological composition injected into the blood should be soluble. Other factors are known in the art, including considerations such as toxicity.

"Systemic administration" means systemic absorption or accumulation of a drug in the bloodstream followed by distribution throughout the body. Routes of administration that result in systemic absorption include, but are not limited to, intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary, and intramuscular.

Examples of agents suitable for formulation with the nucleic acid molecules of the present invention include: P-glycoprotein inhibitors (such as Pluronic P85), which can increase drug entry into the CNS (Jolliet-Riant and Tillement, Fundam. Clin. Pharmacol. 13: 16- 26, 1999); biodegradable polymers such as poly(DL-lactide-glycolide copolymer) microspheres for sustained-release delivery after intracerebral implantation (Emerich, D.F et al., CellTransplant 8: 47-58, 1999, Alkermes, Inc., Cambridge, Mass.); and drug-loaded nanoparticles, such as those made from polybutylcyanoacrylate, which can deliver drugs across the blood-brain barrier and alter neural uptake Mechanism (Prog. Neuropsychopharmacol Biol. Psychiatry 23:941-949, 1999). Other examples of delivery methods for nucleic acid molecules of the invention include materials described in: Boado et al., J. Pharm. Sci. 87:1308-1315, 1998; Tyler et al., FEBS Lett.421:280-284, 1999; Pardridge et al., PNAS USA. 92:5592-5596, 1995; Boado, Adv. Drug Delivery Rev. 15:73-107, 1995; Aldrian-Herrada et al., Nucleic Acid Res. et al., PNAS USA. 96:7053-7058, 1999.

The invention also includes compositions prepared for storage or administration comprising a pharmaceutically effective amount of the desired compound in a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (ed. A. R. Gennaro, 1985). For example, preservatives, stabilizers, dyes and flavor enhancers may be used. These include sodium benzoate, sorbic acid, and parabens. In addition, antioxidants and suspending agents may be used.

A pharmaceutically effective dose is the dose required to prevent, inhibit the occurrence of a disease state, treat or improve the symptoms of a disease state to a certain extent. The dose of active nucleic acid should be 0.01 mg to 50 mg per kg body weight per day.

Aqueous suspensions may contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. The excipients are suspending agents such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum arabic; dispersing or wetting agents can be are naturally occurring phospholipids, such as lecithin, or condensation products of alkylene oxides with fatty acids, such as polyoxyethylene stearate, or condensation products of ethylene oxide with long-chain fatty alcohols, such as heptadecylene Heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitols, such as polyoxyethylene sorbitan monooleate, or ethylene oxide with partial esters derived from fatty acids Condensation products with partial esters of hexitol anhydrides, such as polyethylene sorbitan monooleate. Aqueous suspensions may also contain one or more preservatives, for example ethyl or n-propyl p-hydroxybenzoate, one or more coloring agents, one or more flavor enhancers and one or more Various sweeteners such as sucrose or saccharin.

Oily suspensions may be formulated by suspending the active ingredient in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. Oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening and flavoring agents may be added to provide a palatable oral preparation. These compositions can be preserved by the addition of antioxidants such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Other excipients, for example sweetening, flavoring and coloring agents, may also be present.

The pharmaceutical composition of the present invention may also be in the form of an oil-in-water emulsion. The oily phase may be vegetable oil or mineral oil or a mixture of both. Suitable emulsifiers may be naturally occurring rubbers such as gum arabic or tragacanth, naturally occurring phospholipids such as soy, lecithin, and esters or partial esters derived from fatty acids and hexitols, anhydrides such as sorbitan mono oil esters, and condensation products of said partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.

The pharmaceutical composition may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to methods known in the art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. Additionally, sterile, fixed oils can be employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

siRNA can also be administered in the form of a suppository, eg, for rectal administration of a drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore dissolve in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols.

siRNAs can be extensively modified by using nuclease resistant groups such as 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl, 2′-H modifications, to improve stability. For reviews see Usman and Cedergren, TIBS 17:34, 1992; Usman et al., Nucleic Acid Symp. Ser. 31:163, 1994. siRNA constructs can be purified by gel electrophoresis or by high pressure liquid chromatography using conventional methods and resuspended in water.

Chemically synthesized nucleic acid molecules containing modifications (bases, sugars, and/or phosphates) can protect them from degradation by serum ribonucleases, which can increase their potency. See, for example, Eckstein et al., International Publication No. WO92/07065; Perrault et al., Nature 344:565, 1990; Pieken et al., Science 253,314, 1991; Usman and Cedergren, Trends in Biochem.Sci.17:334, 1992; et al., International Publication No. WO 93/15187; and Rossi et al., International Publication No. WO 91/03162; Sproat, US Patent No. 5,334,711; and Gold et al., US Patent No. 6,300,074. All of the above references describe various chemical modifications that can be made to the base, phosphate and/or sugar entities of the nucleic acid molecules described herein.

There are several examples in the prior art describing sugar, base and phosphate modifications that can be introduced into nucleic acid molecules and that would significantly increase their nuclease stability and efficacy. For example, oligonucleotides can be modified by using nuclease resistant groups such as 2'-amino, 2'-C-allyl, 2'-fluoro, 2'-O-methyl, 2'-O- Allyl, 2'-H nucleotide base modification to improve stability and/or increase biological activity. For reviews, see Usman and Cedergren, TIBS 17:34, 1992; Usman et al., Nucleic Acid Symp. Ser. 31:163, 1994; Burgin et al., Biochemistry 35:14090, 1996. Sugar modification of nucleic acid molecules has been extensively described in the prior art. See Eckstein et al, International Publication PCT No. WO92/07065; Perrault et al, Nature 344:565-568,1990; Pieken et al, Science 253:314-317,1991; Usman and Cedergren, Trends in Biochem.Sci.17:334- 339, 1992; Usman et al., International PCT Publication No. WO 93/15187; Sproat, U.S. Patent No. 5,334,711 and Beigelman et al., J. Biol. Chem. 270:25702, 1995; Beigelman et al., International PCT Publication No. WO97/26270; Beigelman Usman et al., U.S. Patent No. 5,627,053; Woolf et al., International PCT Publication No. WO98/13526; Thompson et al., Karpeisky et al., Tetrahedron Lett. 39:1131, 1998; Earnshaw and Gait, Biopolymers (Nucleic Acid Sciences) 48:39-55, 1998; Verma and Eckstein, Annu.Rev.Biochem.67:99-134, 1998; and Burlina et al., Bioorg.Med.Chem.5:1999-2010, 1997. These publications describe general methods and strategies for identifying sites for introducing sugar, base and/or phosphate modifications, etc., into nucleic acid molecules without modulating catalytic activity. Within these guidelines, similar modifications can be used to modify siRNA nucleic acid molecules of the invention, as described herein, so long as the ability of the siRNA to promote RNAi in cells is not significantly inhibited.

Although chemical modification of oligonucleotide internucleotide linkages containing phosphorothioate, phosphorodithioate, and/or 5′-methylphosphonate linkages improves stability, excessive modification can lead to some Toxicity or reduced activity. Therefore, when designing nucleic acid molecules, the amount of these internucleotide linkages should be minimized. Reducing the concentration of these linkages reduces toxicity, resulting in increased potency and higher specificity for these molecules.

In some embodiments, the invention relates to modified siRNA molecules having a phosphate backbone modification comprising one or more of phosphorothioate, phosphorodithioate, methylphosphonate, phosphotriester , morpholino, amidated carbamate, carboxymethyl, acetamide, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal and/or Alkylsilyl substitution. For a review of oligonucleotide backbone modifications see Hunziker and Leumann, Nucleic Acid Analogues: Synthesis and Properties, in Modern Synthetic Methods, VCH, 1995, pp. 331-417, and Mesmaeker et al., "Novel Backbone Replacements for Oligonucleotides, in Carbohydrate is search Remodifications , "ACS, 1994, pp. 24-39.

Methods of delivering nucleic acid molecules are described in Akhtar et al., Trends Cell Bio. 2: 139, 1992; "Delivery Strategies for Antisense Oligonucleotide Therapeutics," edited by Akhtar, 1995; Maurer et al., Mol. Membr. Biol. 16: 129-140, 1999 ; Hofland and Huang, Handb. Exp. Pharmacol. 137: 165-192, 1999; and Lee et al., ACS Symp. Ser. 752: 184-192, 2000. Beigelman et al., U.S. Patent No. 6,395,713 and Sullivan et al., PCT WO94/02595 further describe general methods for delivering nucleic acid molecules. These guidelines can be used to deliver virtually any nucleic acid molecule. Nucleic acid molecules can be administered to cells by various methods known to those skilled in the art, including, but not limited to, by internal or external encapsulation in liposomes, by iontophoresis, or by introduction to other carriers, such as biodegradable polymers , hydrogels, cyclodextrins (see e.g. Gonzalez et al., Bioconjugate Chem. 10:1068-1074, 1999; Wang et al., International PCT Publication Nos. WO03/47518 and WO03/46185), lactic-co-glycolic acid (PLGA) and PLCA microspheres (see e.g. US Patent No. 6,447,796 and US Patent Application Publication No. US2002130430), biodegradable nanocapsules and bioadhesive microspheres, or via protein carriers (O'Hare and Normand, International PCT Publication No. WO00/53722) . Alternatively, the nucleic acid/vector composition can be delivered locally by direct injection or using an infusion pump. Direct injection of the nucleic acid molecules of the present invention, whether subcutaneously, intramuscularly or intradermally, can be performed using conventional needle and syringe techniques or using needle-free techniques, such as Conry et al., Clin. Cancer Res. 5: 2330-2337, 1999 and Barry et al., International PCT Publication No. WO 99/31262. The molecules of the invention can be used as pharmaceutical formulations. The pharmaceutical formulation prevents, modulates the occurrence of, or treats (reduces symptoms to some degree, preferably all symptoms) a disease state in a subject.

"RNA" refers to a molecule containing at least one ribonucleotide residue. "Ribonucleotide" means a nucleotide having a hydroxyl group at the 2' position of the β-D-ribose-furanose moiety. The term includes double-stranded RNA, single-stranded RNA, isolated RNA, such as partially purified RNA, substantially pure RNA, synthetic RNA, recombinantly produced RNA, and RNA obtained by adding, deleting, substituting and/or changing one or more An altered RNA that differs from the naturally occurring RNA in terms of nucleotides. Such alterations may include, for example, addition of non-nucleotide substances to, for example, the end or interior of the siRNA at one or more nucleotides of the RNA. Nucleotides in the RNA molecules of the present invention also include non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides or deoxynucleotides. These altered RNAs may be referred to as analogs or analogs of naturally occurring RNAs.

"Cap structure" refers to a chemical modification introduced at either end of an oligonucleotide (see, eg, Adamic et al., US Patent No. 5,998,203, incorporated herein by reference). These terminal modifications protect the nucleic acid molecule from exonuclease degradation and facilitate delivery and/or localization within the cell. The cap may be present at the 5'-end (5'-cap) or the 3'-end (3'-cap) or may be present at both ends. In non-limiting examples, 5'-caps include, but are not limited to, glyceryl, inverted deoxyabasic residues (parts); 4',5'-methylene nucleotides; 1-(β-D- Erythrofuranosyl) nucleotides, 4'-thionucleotides; carbocyclic nucleotides; 1,5-anhydrohexitol nucleotides; L-nucleotides; α-nucleotides; bases Modified nucleotides; phosphorodithioate linkages; threo-pentofuranosyl nucleotides; acyclic 3',4'-opened nucleotides; acyclic 3,4-dihydroxybutyl nucleotides ; acyclic 3,5-dihydroxypentyl nucleotide, 3'-3'-inverted nucleotide moiety; 3'-3'-inverted abasic moiety; 3'-2'-inverted core nucleotide moiety; 3'-2'-reverse abasic moiety; 1,4-butanediol phosphate; 3'-phosphoramidate; hexyl phosphate; aminohexyl phosphate; 3'-phosphate; 3 '-phosphorothioate; phosphorodithioate; or a bridged or unbridged methylphosphonate moiety.

Examples of 3'-caps include, but are not limited to, glyceryl, inverted deoxyabasic residues (parts), 4',5'-methylene nucleotides; 1-(β-D-erythrofuranosyl) Nucleotides; 4'-thionucleotides, carbocyclic nucleotides; 5'-amino-alkyl phosphates; 1,3-diamino-2-propyl phosphates; 3-aminopropyl phosphates ;6-aminohexyl phosphate; 1,2-aminododecyl phosphate; hydroxypropyl phosphate; 1,5-anhydrohexitol nucleotide; L-nucleotide; α-nucleotide; Base-modified nucleotides; phosphorodithioate; threo-pentofuranosyl nucleotides; acyclic 3',4'-opened nucleotides; 3,4-dihydroxybutyl nucleotides ; 3,5-dihydroxypentyl nucleotide, 5'-5'-inverted nucleotide moiety; 5'-5'-inverted abasic moiety; 5'-phosphoramidate; 5'-thio Phosphorophosphate; 1,4-butanediol phosphate; 5′-amino; bridged and/or non-bridged 5′-phosphoramidate, phosphorothioate and/or phosphorodithioate, bridged or Unbridged methylphosphonate and 5'-mercapto moieties (see Beaucage and Lyer, Tetrahedron 49:1925, 1993 for a detailed description; incorporated by reference).

The term "non-nucleotide" refers to any group or compound capable of being introduced into a nucleic acid strand in place of one or more nucleotide units (including sugar and/or phosphate substitutions) and allowing the residual base to exhibit its enzymatic activity. The group or compound is abasic in that it does not contain a conventionally recognized nucleotide base such as adenosine, guanine, cytosine, uracil, or thymine, thus missing a base at the 1'-position .

As used herein, "nucleotides" as recognized in the art, include natural bases (standard) and modified bases well known in the art. The base is usually located at the 1' position of the nucleotide sugar moiety. Nucleotides generally include a base, sugar, and phosphate group. Nucleotides may be unmodified or modified in the sugar, phosphate, and/or base moieties, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard Nucleotides and the like; see, e.g., Usman and McSwiggen, supra; Eckstein et al, International PCT Publication No. WO92/07065; Usman et al, International PCT Publication No. WO93/15187; Uhlman & Peyman, supra, which are incorporated herein in their entirety for refer to). Some examples of modified nucleic acid bases known in the art are summarized in Limbach et al., Nucleic Acid Res. 22:2183,1994. Some non-limiting examples of base modifications that can be introduced into nucleic acid molecules include inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxyphenyl , 3-methyluracil, dihydroxyuridine, naphthyl, aminophenyl, 5-alkylcytidine (such as 5-methylcytidine), 5-alkyluridine (such as thymidine), 5-halogenated uridine (such as 5-bromouridine) or 6-azapyrimidine or 6-alkylpyrimidine (such as 6-methyluridine), propyne, etc. (Burgin et al., Biochemistry 35: 14090, 1996 ; Uhlman and Peyman, supra). "Modified base" in this aspect means a nucleotide base other than adenine, guanine, cytosine and uracil or its equivalent at the 1' position.

"Target site" or "target sequence" or "targeted sequence" means a sequence within a target nucleic acid (such as RNA) that is "targeted" by mediated cleavage by an siRNA construct that It contains a sequence complementary to the target sequence in its antisense region.

siRNA molecules can be complexed with DILA2 amino acid compounds or cationic lipids, encapsulated within liposomes, or otherwise delivered to target cells or tissues. The nucleic acid or nucleic acid complex may or may not be incorporated into a biopolymer for local administration by injection, infusion pump or stent. In another embodiment, polyethylene glycol (PEG) can be covalently attached to the siRNA compound or polypeptide or both of the invention. The attached PEG can be of any molecular weight, preferably from about 2,000 to about 50,000 Daltons (Da).

The sense region can be linked to the antisense region by a linker molecule, such as a polynucleotide linker or a non-nucleotide linker.

"Inverted repeat" refers to a nucleic acid sequence containing sense and antisense elements positioned such that they can form a double-stranded siRNA when the repeat is transcribed. The inverted repeat may optionally include a linker or a heterologous sequence, such as a self-cleaving ribozyme between the two elements of the repeat. The inverted repeat elements are of sufficient length to form double-stranded RNA. Typically, each element of the inverted repeat sequence is about 15 to about 100 nucleotides in length, preferably about 20 to 30 bases in nucleotides, preferably about 20 to 25 nucleotides in length, such as 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides in length.

"Nucleic acid" refers to deoxyribonucleotides or ribonucleotides and single- or double-stranded polymers thereof. The term includes nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, synthetic, naturally occurring, and non-naturally occurring, that have binding properties similar to a reference nucleic acid, and that are similar to those of the reference nucleic acid. Nucleotides are metabolized in a similar manner. Examples of such analogs include, but are not limited to, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2'-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).

"Large double stranded RNA" refers to any double stranded RNA that is greater than about 40 bp in size, for example greater than 100 bp or more preferably greater than 300 bp. The sequences of large dsRNAs can represent mRNA fragments or complete mRNAs. The maximum size of a large dsRNA is not limited herein. Double-stranded RNA can include modified bases, where modifications can be made to the sugar phosphate backbone or nucleosides. The modifications may include nitrogen or sulfur heteroatoms or any other modification known in the art.

Double-stranded structures can be formed by self-complementary RNA strands, such as hairpins or microRNAs, or by annealing of two different complementary RNA strands.

"Overlapping" refers to sequences in which two RNA fragments overlap by a number of nucleotides in one strand, for example, where the number of nucleotides (nt) is as few as 2 to 5 nucleotides or 5 to 10 nucleotides sour or more.

"One or more dsRNAs" refers to dsRNAs that differ from each other on the basis of primary sequence.

"Target gene or mRNA" refers to any gene or mRNA of interest. Target genes or mRNAs may include developmental and regulatory genes, as well as metabolic or structural genes or genes encoding enzymes. Target genes can be endogenous or exogenous. The target gene can be expressed in those cells or in the organism whose phenotype is being studied in a manner that directly or indirectly affects the phenotypic characteristic. Such cells include any cell in an adult or embryonic animal or plant, including gametes or any isolated cell, such as exists in an immortal cell line or in primary cell culture.

Applications for Delivery of Active Agents

The compounds and compositions of the invention may be used to deliver any physiologically or biologically active agent, and any combination of active agents, as described above or as known in the art. The active agent may be present in the compositions and uses of the invention in an amount sufficient to provide the desired physiological or ameliorative effect.

The compounds and compositions of the present invention are capable of targeted enhanced delivery of a range of pharmaceutical and biologically active agents in a mammalian subject, including small molecule compounds and drugs, peptides, proteins, antibodies, monoclonal antibodies, antibody-based medicines and vaccines.

Examples of active agents include peptides, proteins, nucleic acids, double-stranded RNA, blood tonics, anti-infectives; antidementias; antivirals, antitumors, antipyretics, analgesics, anti-inflammatory, Allergic agents, antidepressants, psychotropic drugs, cardiotonic agents, antiarrhythmic agents, vasodilators, antihypertensive agents, antihypertensive diuretics, antidiabetic agents, anticoagulants, cholesterol-lowering agents, osteoporosis treatment agents, hormones, antibiotics, vaccines, cytokines, hormones, growth factors, cardiovascular factors, cell adhesion factors, central or peripheral nervous system factors, humoral electrolyte factors, blood organic matter, bone growth factors, gastrointestinal factors, kidney Factors, connective tissue factors, sensory factors, immune system factors, respiratory system factors, reproductive organ factors, androgens, estrogens, prostaglandins, growth hormones, gonadotropins, interleukins, steroids, bacterial toxoids, antibodies, monoclonal Antibodies, polyclonal antibodies, humanized antibodies, antibody fragments and immunoglobulins.

Examples of active agents include erythropoietin, granulocyte colony stimulating factor, insulin, factor VII, factor VIII, factor IX, interferon, heparin, prohirudin, hirulos and hirudin.

Examples of active agents include morphine, hydromorphone, oxymorphone, oxymetholone, allyl levomorphol, codeine, nalmefene, nalofen, naloxone, naltrexone, buprenor morphine, butorphanol or nalbuphine, cortisone, hydrocortisone, fludrocortisone, prednisone, prednisone, methylprednisolone, fludroprednisone, dexamethasone, Betamethasone, paramethasone, fluocinolone, colchicine, acetaminophen, NSAID, acyclovir, ribavirin, trifluridine, arabinofuranosyladenosine (Ara -A), acylguanosine, nordeoxyguanosine, azidothymidine, dideoxyadenosine, dideoxycytidine, spironolactone, testosterone, estradiol, progesterone, gonadotropins, estrogen Hormone, progesterone, papaverine, nitroglycerin, vasodilation intestinal peptide, calcitonin gene-related peptide, cyproheptadine, doxepin, imipramine, cimetidine, dextromethorphan, clozapine, super Oxygen dismutase, neuroneprilysin, amphotericin B, griseofulvin, miconazole, ketoconazole, tioconazole, itraconazole, fluconazole, cephalosporins, tetracyclines, glucosamine Glycosides, erythromycin, gentamicin, polymyxin B, pentafluorouracil, bleomycin, methotrexate, hydroxyurea, dideoxyinosine, floxuridine, 6-mercaptopurine, doxorubicin, Doxor Amycin, nordaunomycin, paclitaxel, paclitaxel, tocopherol, quinidine, prazosin, verapamil, nifedipine, diltiazem, tissue plasminogen activator TPA, epidermis Growth factor EGF, acidic or basic fibroblast growth factor FGF, platelet-derived growth factor PDGF, transforming growth factor TGF-α or β, vasodilation intestinal peptide, tumor necrosis factor TNF, hypothalamic releasing factor, prolactin, prolactin Thyroid hormone TSH, adrenocorticotropic hormone ACTH, parathyroid hormone PTH, follicle-stimulating hormone FSF, luteinizing hormone-releasing hormone LHRH, endorphins, glucagon, calcitonin, oxytocin, carbetocin, aldoetecone, enkephalin, somatostatin, growth hormone, somatomodulin, alpha-melanocyte stimulating hormone, lidocaine, sufentanil, terbutaline, droperidol, scopolamine, gonadotropin-releasing hormone, Ciclopirox, buspirone, cromoglycate sodium, midazolam, cyclosporine, lisinopril, captopril, delapril, ranitidine, famotidine, superoxide Substance dismutase, asparaginase, arginase, arginine deaminase, adenosine deaminase ribonuclease, trypsin, chemical trypsin, papain, bombesin, substance P, vasopressin , α-globulin, transferrin, fibrinogen, β-lipoprotein, β-globulin, prothrombin, ceruloplasmin, α2-glycoprotein α2-globulin, fetuin, α1-lipoprotein , α1-globulin, albumin and prealbumin.

Examples of active agents include opioids or opioid antagonists such as morphine, hydromorphone, oxymorphone, oxymethorphan, allyl levomorphan, codeine, nalmefene, nalorfene, Naloxone, naltrexone, buprenorphine, butorphanol, and nalbuphine; corticosterone, such as cortisone, hydrocortisone, fludrocortisone, prednisone, prednisone, Methylprednisolone, fludroxyprednisolone, dexamethasone, betamethasone, paramethasone, fluocinolone; other anti-inflammatory drugs such as colchicine, ibuprofen, indomethacin, and piroxicam; antiviral drugs, For example, acyclovir, ribavirin, trifluorothymidine, arabinofuranosyladenosine, acylguanosine, nordeoxyguanosine, azidothymidine, dideoxyadenosine, dideoxycytidine; Antiandrogens such as spironolactone; androgens such as testosterone; estrogens such as estradiol; progesterone; muscle relaxants such as papaverine; antihistamines such as cyproheptadine; agents with histamine receptor site blocking activity such as doxepin, imipramine and cimetidine; antitussives such as dextromethorphan; Relaxants such as clozapine; antiarrhythmics; antiepileptics; enzymes such as superoxide dismutase and neuropriphalinase; antifungals such as amphotericin B, griseofulvin, miconazole, Ketoconazole, tioconazole, itraconazole, and fluconazole; antibacterial agents such as penicillins, cephalosporins, tetracyclines, glucosamines, erythromycin, gentamicin, polymyxin B; Cancer agents such as fluorouracil, bleomycin, methotrexate and hydroxyurea, dideoxyinosine, floxuridine, 6-mercaptopurine, doxorubicin, daunorubicin, nordaunomycin, taxol and paclitaxel; antioxidants such as tocopherols, retinoids, carotenoids, ubiquinone, metal chelators and phytic acid; antiarrhythmics such as quinidine; antihypertensives such as prazosin, vitamin lapamil, nifedipine, and diltiazem; analgesics such as acetaminophen and aspirin; monoclonal and polyclonal antibodies, including humanized antibodies and antibody fragments; antisense oligonucleotides; Sexual RNA, interfering RNA, DNA, and viral vectors containing genes encoding therapeutic peptides and proteins.

Compositions and formulations for administration

As used herein, the term "administration" includes all methods of delivering a compound or composition directly and indirectly to the site of action. The compounds and compositions of the invention may be administered alone or in combination with other compounds, compositions or therapeutic agents not disclosed herein.

The compositions and methods of the invention can be administered to a subject by various modes of mucosal administration, including oral, rectal, vaginal, intranasal, intrapulmonary, or transdermal delivery, or by topical delivery to Eyes, ears, skin or other mucous membrane surfaces. In some aspects of the invention, the mucosal tissue layer includes an epithelial cell layer. Epithelial cells may be pulmonary, tracheal, bronchial, alveolar, nasal, oral, epidermal or gastrointestinal. The compositions of the invention may be administered using a propellant, such as a mechanical nebulizer, as well as pressurized, motorized or other types of propellants.

The compositions of the present invention may be administered in aqueous solutions as nasal or pulmonary sprays, and may be dispersed in spray form by various methods well known to those skilled in the art. Pulmonary delivery of the compositions of the invention can be achieved by administering the compositions in the form of liquid droplets, granules or a spray, which can be, for example, aerosolized, nebulized or nebulized. Pulmonary Delivery Compositions may be administered through the nasal or bronchial passages in the form of liquid droplets, granules or spray. Composition granules, sprays or aerosols may be in liquid or solid form. A preferred system suitable for dispersing liquids as a nasal spray is disclosed in US Patent No. 4,511,069. Said formulations may conveniently be prepared by dissolving the composition of the invention in water to prepare an aqueous solution, and sterilizing said solution. The formulations may be presented in multi-dose containers, such as the hermetically sealed dispersion system disclosed in US Patent No. 4,511,069. Other suitable nasal spray delivery systems are described in Transdermal Systemic Medication, Y.W. Chien ed., Elsevier Publishers, New York, 1985; and U.S. Patent No. 4,778,810. Additional aerosol delivery forms may include, for example, compressed air nebulizers, jet nebulizers, ultrasonic nebulizers, and piezoelectric nebulizers, which deliver the bioactive agent dissolved or suspended in a pharmaceutical solvent such as water, ethanol, or mixtures thereof.

Nasal and pulmonary spray solutions of the present invention generally comprise the drug or drug to be delivered, optionally in combination with a surfactant such as a nonionic surfactant (e.g. polysorbate-80) and one or more buffers Prepare together. In certain embodiments of the invention, the nasal spray solution further comprises a propellant. The pH of the nasal spray solution may be about pH 6.8 to 7.2. The pharmaceutical solvent used can also be a slightly acidic aqueous buffer of pH 4 to 6. Other components may be added to enhance or maintain chemical stability, including preservatives, surfactants, dispersants or gases.

In certain embodiments, the invention is a medicament comprising a solution comprising a composition of the invention and a propellant for pulmonary, mucosal or intranasal spray or aerosol.

Dosage forms of the compositions of the present invention may be in the form of liquid droplets or emulsions, or liquids in the form of sprays.

Dosage forms of the compositions of the invention may be solids, which can be reconstituted in liquid prior to administration. Solids can be administered as a powder. Solids may be in capsule, tablet or gel form.

In order to formulate a composition suitable for pulmonary delivery in the present invention, the bioactive agent can be combined with various pharmaceutically acceptable additives or delivery enhancing components and a matrix or carrier suitable for dispersing the active agent. Examples of additives or delivery enhancing components include pH adjusters such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid and mixtures thereof. Other additives or delivery enhancing components include local anesthetics (such as benzyl alcohol), isotonic agents (such as sodium chloride, mannitol, sorbitol), adsorption inhibitors (such as Tween 80), solubilizers (such as cyclodextrin and its derivatives), stabilizers (such as serum albumin) and reducing agents (such as glutathione). When the composition for mucosal delivery is a liquid, as measured by reference to the tonicity of a 0.9% (w/v) physiological saline solution, the tonicity of the formulation is usually adjusted to not cause substantial in the mucosa at the site of administration. irreversible tissue damage. Typically, the tonicity of the solution is adjusted to a value of about 1/3 to 3, more usually 1/2 to 2, most usually 3/4 to 1.7.

The bioactive agent may be dispersed within a matrix or carrier, which may contain a hydrophilic compound capable of dispersing the active agent and any desired additives. The matrix can be selected from a wide range of suitable carriers, including but not limited to polycarboxylic acids or their salts, copolymers of carboxylic anhydrides (such as maleic anhydride) and other monomers (such as methyl (meth)acrylate, acrylic acid, etc.); Hydrophilic vinyl polymers, such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone; cellulose derivatives, such as hydroxymethyl cellulose, hydroxypropyl cellulose, etc.; and natural polymers, such as chitosan, collagen , sodium alginate, gelatin, hyaluronic acid and their non-toxic metal salts. Biodegradable polymers can be selected as the matrix or carrier, such as polylactic acid, poly(lactic-co-glycolic acid), polyhydroxybutyric acid, poly(hydroxybutyric-co-glycolic acid), and mixtures thereof. Synthetic fatty acid esters, such as polyglycerol fatty acid esters, sucrose fatty acid sugar esters, etc., can be used as carriers. Hydrophilic polymers and other supports, either alone or in combination, can impart increased structural integrity to the support through partial crystallization, ionic bonding, cross-linking, and the like. The provided carrier may be in a variety of forms, including fluid or viscous solutions, gels, patches, powders, microspheres, and films for direct application to the nasal mucosa. The use of selected carriers according to the invention can facilitate the absorption of bioactive agents.

The bioactive agent can be combined with the matrix or carrier in various ways, and the release of the active agent can be by diffusion, carrier breakdown or related water channel formulation. In some cases, the active agent is dispersed in microcapsules (microspheres) prepared from a suitable polymer such as isobutyl 2-cyanoacrylate (see, e.g., Michael et al., J. Pharmacy Pharmacol. 43:1-5, 1991). ) or nanocapsules (nanospheres), and dispersed in a biocompatible dispersion medium applied to the nasal mucosa, which results in sustained delivery and bioactivity over an extended period of time.

Formulations for mucosal, nasal or pulmonary delivery may include hydrophilic low molecular weight compounds as bases or excipients. The hydrophilic low molecular weight compound provides a channel medium through which a water-soluble active agent such as a physiologically active peptide or protein can diffuse through the matrix to the body surface that absorbs the active agent. The hydrophilic low molecular weight compound optionally absorbs water from the mucosa or environment of administration and solubilizes the water-soluble active peptide. The molecular weight of the hydrophilic low molecular weight compound is usually not more than 10,000, and preferably not more than 3000. Examples of hydrophilic low molecular weight compounds include polyol compounds such as oligosaccharides, disaccharides and monosaccharides including sucrose, mannitol, lactose, L-arabinose, D-erythrose, D-ribose, D-xylose , D-mannose, D-galactose, lactulose, cellobiose, gentiobiose, glycerin, polyethylene glycol, and mixtures thereof. Examples of further hydrophilic low-molecular weight compounds include N-methylpyrrolidone, alcohols (such as oligvinyl alcohol, ethanol, ethylene glycol, propylene glycol, etc.) and mixtures thereof.

The compositions of the present invention optionally include pharmaceutically acceptable carrier substances that approximate the desired physiological conditions, such as pH adjusters and buffers, tonicity adjusters, and wetting agents, such as sodium acetate, sodium lactate, sodium chloride, Potassium Chloride, Calcium Chloride, Sorbitan Monolaurate, Triethanolamine Oleate, and mixtures thereof. For solid compositions, nontoxic pharmaceutically acceptable carriers can be used, which include, for example, pharmaceutically graded mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate, etc. .

In certain embodiments of the invention, the bioactive agent may be administered in a time release formulation, for example, in a composition comprising a slow release polymer. The active agent can be prepared using a rapid release resistant carrier, such as a controlled release carrier such as a polymer, a microencapsulated delivery system or a bioadhesive glue. In the various compositions of the invention, prolonged delivery of the active agent can be achieved approximately by including in the composition an agent which delays absorption, for example aluminum monostearate hydrogel and gelatin.

In certain embodiments of the invention, the compositions may include one or more natural or synthetic surfactants. Certain natural surfactants can be found in the human lungs (pulmonary surfactants) and are complex mixtures of phospholipids and proteins that form a monolayer on the air-liquid surface of the alveoli, reducing the surface tension during exhalation to approximately 0, and prevent alveolar rupture. More than 90% by weight of lung surfactant is composed of phospholipids, of which approximately 40 to 80% is DPPC, and the remainder is the unsaturated lecithins POPG, POPC and phosphatidylglycerols. The remaining 10% by weight of surfactant consists of plasma proteins and apoproteins such as surface protein (SP)-A, SP-B, SP-C and SP-D.

Examples of natural surfactants that can be used in the present invention include SURVANTA ^™ (belacontan), CUROSURF ^™ (poractantalfa) and INFASURF ^™ (calfactant) and mixtures thereof.

Examples of synthetic surfactants include: cinnaptide; combinations of dipalmitoyl lecithin, palmitoyl oleoyl phosphatidyl glycerol and palmitic acid; SURFAXIN ^™ (lucinactant); and EXOSURF ^™ (colfosceril); Components of butylphenol aldehyde, DPPC, and cetyl alcohol; and mixtures thereof.

Methods for preparing the delivery composition include ethanol injection and extrusion using the Northern Lipids Lipex Extruder system packed with a polycarbonate membrane filter of defined pore size. Uniformly sized particles can be obtained using sonication using a probe tip and bath sonicator. Homogeneous and monodisperse particle sizes can be achieved without the addition of nucleic acid components. For in vitro transfection compositions, the nucleic acid component can be added after the transfection agent is formulated and stabilized with buffer components. For in vivo delivery compositions, the nucleic acid component is part of the formulation.

The compositions and formulations of the invention may be administered by various routes, eg, to achieve systemic delivery via intravenous, parenteral or intraperitoneal routes. In certain embodiments, the formulation can be delivered intracellularly, eg, in cells of a target tissue, such as the lung or liver, or in an inflammatory tissue. The invention includes compositions and methods for delivering formulations by removing cells from a subject, delivering an agent into the removed cells, and reintroducing the cells into the subject. In certain embodiments, the present invention provides methods of delivering formulations in vivo. The composition can be administered to the subject intravenously, subcutaneously or intraperitoneally. In certain embodiments, the present invention provides methods for in vivo delivery of formulations to the lungs of a mammalian patient.

The active agent liposome compositions of the present invention can be used in pharmaceutical compositions for use in vivo. Administration of the active agent liposomal compositions of the present invention to a subject may be by parenteral, oral, by inhalation, topical, mucosal, rectal or buccal routes. Parenteral use includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrastemal, intrathecal, intralesional, intracranial injection or infusion techniques.

An effective amount of the active agent liposome composition of the present invention for treating a particular disease is usually an amount sufficient to ameliorate or alleviate the symptoms of the disease. The composition may be administered as a single dose, or may be administered in repeated doses.

other implementations

All publications, references, patents, patent publications and patent applications cited herein are expressly incorporated by reference in their entirety.

While a number of embodiments have been described herein, and numerous details are provided for purposes of illustration, it will be apparent to those skilled in the art that the invention encompasses additional embodiments, and that certain details described herein may be suitably incorporated without departing from this disclosure. The circumstances of the invention change. The invention includes such additional embodiments, modifications and equivalents. In particular, the invention includes any combination of features, terms or elements of the various illustrative components and examples.

As used herein, the term "a" and similar terms in the present description and claims are to be construed as including the singular and the plural.

The terms "comprising", "having", "including" and "" are to be interpreted as open-ended terms meaning, for example, "including but not limited to". Accordingly, terms such as "comprising", "having", "including" and "comprising" are to be interpreted inclusively and not exclusively.

The recitation of a range of values herein refers to each individual value and any discrete value falling within the range, as if it were individually recited herein, regardless of whether certain values in the range were expressly recited or not. For example, the range "4 to 12" includes, without limitation, the values 5, 5.1, 5.35, and any other integer, fraction, or rational number greater than or equal to 4 and less than or equal to 12. It should be understood that the specific values used herein are exemplary and do not limit the scope of the invention.

The recitation of a number of carbon atoms in a range herein refers to each individual value and any discrete value falling within that range, as if it were individually recited herein, regardless of whether certain values within that range are expressly stated. For example, the term "C1-22" includes, but is not limited to, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, C15, C16, C17, C18, C19, C20, C21 and C22.

The definitions of technical terms provided herein should be construed to include unexplained meanings related to terms known to those skilled in the art, and are not intended to limit the scope of the present invention. Definitions of technical terms provided herein should be construed as superior to, or incorporated by reference herein, alternative definitions in the art to the extent that alternative definitions conflict with definitions provided herein.

The examples given herein, and the exemplary language used herein, are for purposes of explanation only and are not intended to limit the scope of the invention.

Where a list of examples is given, such as a list of compounds or molecules suitable for the invention, it will be apparent to those skilled in the art that mixtures of the listed compounds or molecules are also suitable.

Example

Example 1

Method for preparing liposome formulations containing RNA

This example describes an embodiment of a method for preparing an RNA-containing liposome formulation. Some of the materials used in the method are summarized below:

C18:1-n-Arg-C16 (palmitoyl oleoyl n-arginine, PONA) (MDRNA, Inc.) (chemical formula weight 683.3)

1,2-Dimyristoyl-tin-glyceryl-3-phosphatidylethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt) (DMPE-PEG2k) (Genzyme Pharmaceuticals, Cambridge, Mass.)

Cholesterol (Solvay Pharmaceuticals)

Cholesterol Hemisuccinate (CHEMS) GMP (Merck Eprova AG)

Ethanol (pure, 200 proof); sterile water for injection

Sodium Phosphate: Monobasic, Anhydrous, Dibasic, Anhydrous

Sucrose, 99+%

5N sodium hydroxide; 2N hydrochloric acid; glacial acetic acid

Triethanolamine (Tris) USP grade (Research Organics)

0.2μm filter bottle, 150mL capacity, PES

Calibrated 20µL, 200µL and 1mL Rainin Pipettes

Iso-disc filter PTFE25-10

Cole-Parmer Online Static Mixer

Watson Marlow 520Di pump; Watson Marlow 523 pump; Filtertec pump

Vivaflow 50 100,00MWCO PES(Sartorius)

Slide-a-Lyser Dialysis Cassette 10,000MWCO (Pierce)

Prepare buffer solution Sucrose phosphate (SUP) formulation buffer (20 mM sodium phosphate, 215 mM sucrose, pH 7.4) as follows. Add 2.17 g of anhydrous monobasic sodium phosphate and 8.79 g of anhydrous disodium phosphate to a graduated cylinder containing 3600 mL of Milli-Q DI water and mix thoroughly with a stir bar. The pH was adjusted to pH 7.4 with 5N sodium hydroxide or 2N hydrochloric acid. 294.38g of sucrose was slowly added and fully dissolved. Adjust the final water volume to 4L. The solution was filtered using a 0.2 μm filter.

Follow the steps below to prepare USP stock solutions containing 25 mM liposome-forming molecules in 90% v/v ethanol. Dispense 90 mL of ethanol USP (200 proof) in a clean autoclaved 100 mL Pyrex bottle. 1291umol C18:1-n-Arg-C16 (PONA), 721.6umol cholesterol hemisuccinate (CHEMS) powder, 61.7umol DMPE-PEG2K powder and 515umol cholesterol were continuously added to ethanol. The components were all added to the solution and mixed well with a stir bar. The mixture was sonicated for 15 min. Add 10 mL of Sterile Water for Injection USP and mix well. The stock solution was filtered through a 1 um pore size ISO-DISC filter PTFE-25 mm. Stock solutions were stored at 80°C and analyzed for DILA2 amino acid compounds and lipid fractions by reverse phase HPLC with evaporative light scattering detection.

Follow the steps below to prepare siRNA stock solutions in sterile water for injection. Dispense 5 mL of Sterile Water for Injection into a sterile 15 mL Falcon tube. Add 100 mg siRNA powder to the tube and vortex well. The solution was filtered through a 0.22 uM Millex GP filter unit using a 10 mL syringe. siRNA solutions were stored at -20°C and diluted 1:1000 to check purity and concentration by OD (A260 and A280).

A Watson Marlow 520Di peristaltic pump was calibrated at a flow rate of 40 mL/min. The pump was set to 210 rpm and disconnected from the tubing. Flush the lines by pumping through 40 mL of 90% ethanol. Ethanol was pumped into the beaker for 15 seconds and weighed to determine the flow rate (mL/min). Adjust the pump speed to provide a flow rate of 40 ± 0.5 mL/min. Pumps for siRNA and sucrose phosphate solutions were calibrated in a similar manner.

siRNA formulations were prepared using the three solutions as follows. (a) The first solution for pumping is siRNA solution. Prepare the first solution by diluting siRNA with SUP buffer in a 50 mL conical tube and vortexing well. (b) The second solution used for pumping is a solution of DILA2 amino acid compound and three lipids. A 90% ethanol stock solution of mixed lipids containing the following lipids was prepared: CHEMS, cholesterol and DMPE-PEG. . To the lipid stock solution was added the DILA2 amino acid compound. To the lipid stock solution, a Tris-containing sterile aqueous solution for injection was added to form a 1:1 molar Tris:CHEMS concentration in the solution. Prepare a second solution for pumping by pipetting the mixed lipid stock solution into a 50 mL conical tube with a positive displacement pipette, diluting with 90% ethanol and vortexing well. (c) The third solution used for pumping is SUP buffer solution.

Follow the steps below to prepare siRNA preparations. The first siRNA solution and the second liposome-forming molecule solution are simultaneously pumped into the impingement fluid. The first 1 mL of impingement fluid was discarded and the siRNA preparation was collected in a vessel. The SUP buffer solution was pumped into the vessel using a Watson Marlow 323 pump to adjust the concentration of ethanol to approximately 33%. The siRNA preparations were incubated in the dish for 1 hour with gentle shaking on a plate magnetic stirrer.

After incubation, formulations were loaded into Pierce slide-a-lyzer dialysis cassettes with 10,000 MWCO and dialyzed against 100 volumes of SUP for 12-18 hours at 4°C.

This example further describes an embodiment of the method of preparing RNA-containing liposome formulations by tangential flow and diafiltration. siRNA formulations were provided as above, except that the final dialysis step was replaced by a tangential flow filtration (TFF) process.

The siRNA preparations were diluted to a final concentration of 10% (v/v) ethanol over 2 minutes with gentle shaking on a plate magnetic stirrer.

The TFF system using the Sartorius Vivaflow 50 100,000 MWCOPES membrane was rinsed with 50 mL of 70% ethanol USP and then recirculated with 100 mL of 70% ethanol at a pump flow rate of 60 mL/min. Rinse the TFF system with 50 mL of sterile water, then recirculate with 100 mL of sterile water at a pump flow rate of 60 mL/min. Purge the TFF system with 50 mL of SUP, then recirculate with 100 mL of SUP at a pump flow rate of 60 mL/min.

Diluted siRNA preparations were loaded into TFF vessels and concentrated 5 times to a final siRNA concentration of 0.5 mg/mL (feed pressure -20 psi, retentate pressure <0.2 psi, permeate flow rate -2 mL/min). The maximum amount of siRNA formulated in liposome compositions treated was 1 mg per ^cm2 of membrane.

Concentrated siRNA preparations were filtered through diafiltration through 5 volumes of SUP (where ethanol had been removed) at a flow rate of 2 mL/min.

The concentrated siRNA preparation was further concentrated to the desired volume (1 mg/ml siRNA).

This example further describes an embodiment of a method of preparing an RNA-containing liposome formulation by sterile filtration of an siRNA liposome formulation. siRNA formulations were provided as described above. Draw 10 mL of siRNA formulation into a 10 mL polypropylene syringe and remove air bubbles. The siRNA preparation was filtered through a 0.22uM Millex GP filter unit. Moderate pressure was applied to the syringe to filter 10 mg of the siRNA preparation (1 mg siRNA/mL) through the Millex GP filter unit. A 1 mL sample of the drug product was stored at 80°C in a 3 mL Type I sterile glass tube for later use.

Example 2

siRNA liposome formulation

Embodiments of liposomal siRNA formulations of the present invention are shown in Table 6.

Table 6: Liposome siRNA formulations

component μM MW mg/ml Administration (mg/kg) dsRNA 7.5 13255.4 0.100 1.0 DMPE-PEG2K 38.5 2815 0.108 1.1 chol 366.2 386 0.141 1.4 CHEMS 506.8 486 0.246 2.5 PONA 929.3 683 0.635 6.3

Example 3

Effect of Physical Process Parameters on RNA-Containing Liposome Compositions

In this example, the effect of certain process parameters for collection, incubation and quenching on the properties of siRNA liposome compositions was observed. Compositions were prepared by using the basic method described in Example 1.

In various embodiments, the active agent of the composition is dsRNA for silencing ApoB. The liposome forming components are DILA2-containing amino acid compound C18:1-normal Arg(NH ₃ Cl)-C16 and lipid cholesterol hemisuccinate (CHEMS, Anatrace, CH210), cholesterol (Anatrace CH200) and DMPE- Ethanol-water solution of PEG2k (Genzyme).

In the first embodiment, as shown in Table 7, the effect of the concentration of the organic solvent in the collection step on the particle size and dispersibility of liposomes was observed. Calculate the concentration of organic solvent ethanol based on the flow rate and transfer tube diameter. The incubation time of each formulation in Table 7 is 4 hours.

Table 7: Collection and incubation of liposomal siRNA formulations

preparation pH Z-avg PdI Avg pack Collected in 33% EtOH 7.4 152 0.11 89% Collected in 37% EtOH 7.4 161 0.12 89% Collected in 40% EtOH 7.4 242 0.30 89%

The results in Table 7 show that, generally speaking, the size of liposome particles increases with the increase in the concentration of the organic solvent ethanol in the collector. The dispersibility of particle size distribution also increases with the increase of organic solvent concentration. The results in Table 7 demonstrate that high levels of encapsulation of active siRNA agents were achieved using the liposome composition prepared from the impingement fluid and the mixture in the collection vessel (pH 7.4).

In a second example, the effect of incubation time on the gene silencing activity of liposomal siRNA formulations in mice was observed. The in vivo ApoB gene silencing activity of liposomal formulations was determined in mouse liver. Some RNAi agents for silencing ApoB are described in WO08/109357.

The in vivo ApoB gene silencing activity of certain liposome formulations was determined in mouse liver and compared to mouse serum cholesterol levels. ApoB mRNA lowering activity in vivo and the corresponding lowering of serum cholesterol in vivo are shown in Table 8. Each liposome formulation in Table 8 was [C18-n-Arg-C16/CHEMS/chol/DMPE-PEG2k (50/28/20/2)]. The dose in each case was 1.0 mg/kg/day. Each formulation was prepared using an ethanol concentration of 33% based on flow rate and transfer tube diameter in the collection vessel.

Table 8: Effect of incubation time on gene silencing activity in mice

Incubation time (hr) In vivo ApoB knockdown (%) Reduction of serum cholesterol (%) 0 19 8 1 31 twenty four 2 38 6 4 51 27

The results in Table 8 show that the in vivo gene silencing knockdown activity of the liposome formulation containing the ApoB gene silencing RNAi reagent increases to a favorable level as the incubation time increases.

In the third example, as shown in Table 9, the effect of incubation time on the gene silencing activity of liposomal siRNA formulations in vivo was observed. In these experiments, liposomal siRNA formulations were prepared using dialysis rather than TFF filtration. The compositions shown were prepared using impingement fluid and the mixture (pH 7.4) in the collection vessel. As shown in Table 9, the concentration of the organic solvent ethanol in the collection vessel varied from 30-36%. Each liposome formulation in Table 9 was [C18-n-Arg-C16/CHEMS/chol/DMPE-PEG2k (50/28/20/2)].

Table 9: Effects of incubation under various concentrations of ethanol in the collection container on the in vivo gene silencing activity of liposomal siRNA preparations

The results in Table 9 show that the gene silencing activity of the liposome formulation containing the ApoB gene silencing RNAi agent in mice was significantly increased to a favorable level when the incubation time was used.

In a fourth example, as shown in Table 10, the effect of quenching liposomal siRNA formulations on their gene silencing activity in mice was observed. In these experiments, liposomal siRNA formulations were prepared using the impingement fluid and the mixture in the collection vessel (pH 7.4) and an incubation time of 1 hour. As shown in Table 10, the concentration of the organic solvent ethanol in the collection vessel was quenched from 33% to a reduced concentration. Each liposome formulation in Table 10 is [C18-n-Arg-C16/CHEMS/chol/DMPE-PEG2k]. The stability of the indicated formulations was determined by calculating the average particle size and entrapment of siRNA active agents at 1 hour and 48 hours after quenching.

Table 10: Quenching of liposomal siRNA formulations

The results in Table 10 demonstrate that liposome formulations containing RNAi agents maintained a stable average particle size and high level of encapsulation of RNAi agents for a period of 48 hours after quenching to ethanol concentrations below about 25%.

Example 4

Effect of pH on liposome compositions prepared by incubation

In this example, the effect of pH on the preparation of liposome compositions was observed. Compositions were prepared using the basic method described in Example 1 by impact and incubation.

The active agent shown is 1 mg/mL of ApoB-silencing dsRNA prepared in aqueous solution.

The liposome forming components are DILA2-containing amino acid compound C18:1-normal Arg(NH ₃ Cl)-C16 and lipid cholesterol hemisuccinate (CHEMS, Anatrace, CH210), cholesterol (Anatrace CH200) and DMPE- Ethanol solution of PEG2k (Genzyme). The relative amounts of DILA2 amino acid compounds and lipids are (50/28/20/2), which represent the components used in the indicated compositions (C18:1-n-Arg( _NH3Cl )-C16/CHEMS/Cholesterol /DMPE-PEG2k) relative to the total amount of DILA2 amino acid compound plus lipid (w/w).

As shown in Table 11, dsRNA formulations with a Z-average particle size of 128-137 nm were prepared at pH 7.4 and pH 4. The formulations in Table 11 were prepared by adding buffer to the impingement fluid to adjust the concentration of ethanol to about 33%, followed by turbulent mixing. Fluid was collected, and the collected mixture was incubated for 1 hour.

Table 11 : Liposome compositions at pH 7.4 and 4

In conclusion, the results shown in Table 11 and the results shown in Tables 7, 9 and 10 of Example 3 indicate that a formulation of liposome-encapsulated RNAi-inducing agent was prepared at a pH of 7.4.

Example 5

Preparation of RNA-containing liposome compositions by flow rate control

In this example, the liposome composition was prepared by controlling the impingement fluid composition using the flow rate of the RNAi agent solution and the solution of the liposome-forming components. Compositions were prepared by impact and incubation using the basic method described in Example 1, except that the flow rates of the RNAi reagent solution and the liposome-forming component solution were adjusted to obtain organic A certain concentration of solvent and RNAi-agent. The formulations indicated were prepared using impingement fluid collected and incubated for 1 hour. The pH is 7.4 and the active agent is dsRNA for ApoB silencing.

The liposome forming components are DILA2-containing amino acid compound C18:1-normal Arg(NH ₃ Cl)-C16 and lipid cholesterol hemisuccinate (CHEMS, Anatrace, CH210), cholesterol (Anatrace CH200) and DMPE- Ethanol solution of PEG2k (Genzyme). The relative amounts of DILA2 amino acid compound and lipid were (50/28/20/2).

As shown in Table 12, dsRNA formulations were prepared using a flow rate ratio of RNAi reagent solution to liposome-forming component solution of 1.7:1, 3:1, and 5:1.

Table 12: Preparation of liposome compositions with controlled flow rate ratios

The results in Table 12 show that using a ratio of the flow rate of the RNAi reagent solution to the flow rate of the liposome-forming component solution of about 2 to about 5 produced good encapsulation of the active agent and gene silencing activity for ApoB in mice. liposome composition.

The results in Table 12 show that average particle sizes as low as 80 nm and low particle size dispersity were achieved with a ratio of the flow rate of the RNAi agent solution to the flow rate of the liposome-forming component solution of about 3 to about 5.

Example 6

Filtration of RNA-containing liposome compositions

In this example, the effect of concentrating active RNAi agents by tangential flow filtration in the preparation of liposome compositions was observed. Compositions at pH 7.4 were prepared using the basic method described in Example 1 by collecting the impingement fluid under 22% EtOH in a collection vessel and incubating for 30 minutes. The composition was quenched to 10% EtOH concentration for tangential flow filtration.

The liposome formation component is containing DILA2 amino acid compound C18:1-normal Arg-C16 and lipid cholesterol hemisuccinate (CHEMS, Anatrace, CH210), cholesterol (Anatrace CH200) and DMPE-PEG2k (Genzyme) weak. The relative amounts of DILA2 amino acid compound and lipid were (50/28/20/2).

Formulations were filtered by tangential flow filtration using Amersham PES columns. As shown in Table 13, under tangential flow filtration performed to concentrate the concentration of the active RNAi agent up to 16-fold, the compositions shown maintained a stable relative particle size and encapsulation of the active RNAi agent. The final concentration of active RNAi reagent was up to 5mg/ml.

Table 13: In vivo gene silencing activity of liposomal RNAi formulations prepared by incubation and filtration

Example 7

Stability of RNA-containing liposome compositions

In this example, the stability of the liposome composition at elevated temperature for 7 days was observed. Compositions were prepared using the basic method described in Example 1 by impact and incubation for 1 hour. A turbulent mixing tube was used and the concentration of EtOH in the collection vessel was 33%. After preparation, the formulation was kept at 45°C for 7 days. After 7 days, the average particle size was 116 nm and the encapsulation was 71%. For the heat-treated formulation, no loss of ApoB gene silencing activity in mice was observed after 7 days.

Example 8

peptide binding region

The relative strength of binding of cationic peptides to RNAi-inducing agents was determined using a dye binding assay.

RNAi inducer (10 ml) was prepared in 7.8 μl to make a stock solution of 20 μg/ml, then 75 μl/well. SYBR-Gold dilutions (15ml) were prepared in 3.75[mu]l, a 1:4000 dilution for the 2.5X stock.

Peptides were dissolved and diluted in Hepes buffer containing 5% glucose. The peptides were further diluted so that 75 [mu]l could be added to each well to form the desired N:P (range 0-4). The peptide is believed to be 50% pure, but the actual peptide amount is unknown.

A SYBR-Gold dye binding assay was performed. 96-well plate assays were performed using a sample volume of 150 μl per well. The final concentration of dsRNA was 10 μg/ml in 10 mM hepes/5% glucose, pH 7.4. Peptides were diluted into different working solutions so equal volumes were added to achieve different N/P ratios. For the addition process, dsRNA (75 μl, 20 μg/ml) was added first followed by 150 μl 2.5×SYBR-Gold. Peptide (75 [mu]l) was then added to compete for the SYBR dye. The total volume is 300 μl. Fluorescence was corrected for the background of the dye alone in the buffer. SYBR-Gold ex/em read at 495nm/537nm on a Molecular Devices plate reader.

Formulation particle size was determined by transfer to a 384-well plate for measurement using a Wyatt particle sizer. Each well of the transferred 96-well plate was performed in duplicate. The volume held in the plate was 200 [mu]l.

When appropriate, release of the peptide is triggered by disulfide bond reduction or enzymatic cleavage. Cysteine-terminated peptides can be cleaved by glutathione reduction. V-Cit-containing peptides can be cleaved by cathepsin B enzymatic cleavage. Glutathione is present in cells at 0.1 to 10 mM, and cathepsin B is present in lysosomes at 1 mM. (For cathepsin B at 0.14 ng/μl see BMC Gastroenterology 2002, 2:16).

For release, the appropriate molecule was added to one of duplicate wells at a final concentration of 1 mM and SYBR GOLD fluorescence was measured over time.

As shown in Figure 6, the binding of the polyarginine binding region to dsRNA increases with the length of the polyarginine binding region. In Figure 6, the strongest binding (best ability to displace SYBR-Gold dye) was observed with PN3499, peptide (SEQ ID NO: 353) RRRRRCCRRRRR (dimeric peptide containing 10 arginines in total).

Example 9

In vitro assay of PPIB gene expression knockdown in A549 cells

The cyclophilin B (PPIB) gene knockdown assay can be used as an in vitro test of the primary activity of an interfering RNA delivery formulation. In general, assays were performed as described below with minor variations.

Cyclophilin B (PPIB) gene expression knockdown was determined in human alveolar epithelial basal cells A549. For PPIB gene knockdown assays, A549 cells were transfected with interfering RNA preparations, total RNA was prepared 24 hours after transfection, and PPIB mRNA was analyzed by RT-PCR. QRT-PCR for 36B4 (acid ribosomal phosphoprotein PO) mRNA expression was performed for normalization.

A549 cells were seeded at 7,500 cells/well (96 wells) and cultured overnight in medium. The confluence at the time of transfection was about 50%. The transfection complex was prepared by adding the interfering RNA to the medium (OptiMEM ^TM ) and vortexing, adding the delivery formulation to the medium (OptiMEM ^TM ) and vortexing, and finally mixing the interfering RNA in the medium with The delivery formulations in the medium were mixed and incubated at room temperature for 20 minutes to obtain transfection complexes. The medium in which the cells were incubated was replaced with fresh OptiMEM ^™ , and the transfection complex was added to each well. Cells were incubated at 37°C and 5% _CO2 for 5 hours, then complete medium was added (to a final fetal bovine serum concentration of 10%) and incubation was continued until 24 hours post-transfection.

For PPIB gene knockdown, cells were lysed and RNA was prepared (Invisorb RNA Cell HTS96-Kit/C, Invitek, Berlin, or RNeasy 96 Kit, Qiagen). Quantitative RT-PCR was performed using a one-step qRT-PCR kit (Invitrogen) on a DNA Engine Opticon2 thermal cycler (BioRad).

The primers used by PPIB are:

(SEQ ID NO: 354)

5'-GGCTCCCAGTTTCTTCATCAC-3' (forward) and

(SEQ ID NO: 355)

5'-CCTTCCGCACCACCTC-3' (reverse) and

(SEQ ID NO: 356)

5'-FAM-CTAGATGGCAAGCATGTGGTGTTTGG-TAMRA-3' was used as a probe.

For 36B4, the primers are:

(SEQ ID NO: 357)

5'-TCTATCATCAACGGGTACAAACGA-3' (forward) and

(SEQ ID NO: 358)

5'-CTTTTCAGCAAGTGGGAAGGTG-3' (reverse) and

(SEQ ID NO: 359)

5'-FAM-CCTGGCCTTGTCTGTGGAGACGGATTA-TAMRA-3' was used as a probe.

The structures of some double-stranded RNAs (dsRNAs) of the present invention are shown in Table 14.

Table 14: Double-stranded RNA

In Table 14, "mU" represents 2'-O-methyluridine, "mC" represents 2'-O-methylcytidine, and "s" represents a phosphorothioate bond.

Example 10

Knockdown of PPIB gene expression using layered vectors and triggered release peptides

Nanoparticle carriers of RNAi-inducing agents were assayed for PPIB gene knockdown activity in A549 cells. A binary complex of the dsRNA RNAi inducer and the triggered release peptide initially forms at a specific N/P ratio. Endolysosomal reagent was added, which adjusted the N/P ratio to the final value.

Layered carrier formulations are typically prepared by first vortexing the dsRNA in HEPES/dextrose buffer. The triggered release peptide is added under vortexing to complex with the dsRNA. The complexes were incubated for 15 minutes. Glutaraldehyde was added and the core was allowed to crosslink for 1.5 hours. The reaction was quenched by the addition of 1M Tris buffer, pH 7.4. Endolysosomal reagent was added and the vector mixture was incubated for 15 minutes before adding to the cells.

PPIB gene expression knockdown assays using layered vectors containing triggered release peptides are shown in Table 15. The results in Table 15 demonstrate that vectors containing triggered release peptides can effectively deliver active dsRNA agents to cells in the presence of endolysosomal agents to produce significant gene silencing.

Table 15: Knockdown of PPIB gene expression using triggered release peptides

The materials used in this example are as follows:

PN4110

SEQ ID NO: 373

WWHHKKRRCCRRKKHHWW

PN3033 (diINF7)

SEQ ID NO: 374

NH ₂ -GLFEAIEGFIENGWEGMIDGWYGC-CO ₂ H

The effect of the final N/P ratio on the knockdown assay of PPIB gene expression using layered vectors containing triggered release peptides is shown in Table 16. The results in Table 16 demonstrate that, in the presence of endolysosomal agents, vectors containing triggered release peptides can effectively deliver active dsRNA agents to cells to produce significant gene silencing. In addition, the results in Table 16 also show that at lower final N/P ratios of 2.5-3.5, the knockdown of the layered carrier containing the triggered release peptide is enhanced in vitro.

Table 16: Effect of Final N/P Ratio on Gene Knockdown in Vitro

Example 11

Carrier particles with an advantageous low delivery efficiency ratio

A batch of carrier nanoparticles was prepared using DX227 condensed with PN4110. The delivery efficiency ratio for this batch was 0.63. The particle diameter is 223nm (Z-avg, PDI 0.2).

A batch of carrier nanoparticles was prepared using DX4227 condensed with PN183. The delivery efficiency ratio for this batch was 1.28. The particle diameter is 208nm (Z-avg, PDI 0.2).

This example uses the following substances:

PN183

SEQ ID NO: 375

NH ₂ -KETWWETWWTEWSQPGRKKRRQRRRPPQ

Example 12

Liposome formulations prepared from amino acid lipids loaded with carrier particles

Liposome formulations of RNAi agents and amino acid lipids were prepared using the compositions shown in Table 17. RNAi agents against ApoB are described in WO08/109357.

Table 17: Liposome formulations of ApoB RNAi reagents and amino acid lipids

Example 13

Liposome formulations prepared from amino acid lipids loaded with peptide condensate carrier particles in vitro Knockdown of ApoB gene silencing in HepG2 cells

The in vitro ApoB gene silencing activity of liposome formulations prepared from amino acid lipids loaded with peptide condensate carrier particles was determined. ApoB gene knockdown activity was obtained from in vitro experiments in HepG cells. Normalized ApoB mRNA expression values were calculated for the indicated preparations.

The method and protocol used for the HepG assay are as follows:

Day 1: Add 25 μL of complexes to wells, then add 75 μL of cells to wells in DMEM containing 10% FBS or serum-free OPTIMEM. For serum-free OPTIMEM, add 100 μL of complete medium with 20% serum after 4 to 5 hours to make a final FBS concentration of 10%.

Day 2: Cells were lysed at 24 hours, RNA was prepared, and qRT-PCR was performed for ApoB and 36B4, or qRT-PCR for GAPDH mRNA at day 3.

Preparation of liposomal formulation [C18:1-n-Arg-C16/CHEMS/chol/DMPE-PEG2k(50/32/16/2)], wherein C18:1-n-Arg-C16 is in U.S. Patent Application 12/114,284 described amino acid lipids. The indicated liposome formulations were loaded with peptide condensate carrier particles DX4227/PN4110. The initial N/P ratio was 0.8. This formulation showed a 91% knockdown for 100 nM DX4227 RNAi reagent compared to Qneg.

Additional liposome formulations [C18:1-n-Arg-C16/CHEMS/chol/DMPE-PEG2k (50/32/16/2)] were prepared and loaded with peptide condensate carrier particles as shown in Table 18 DX4227/PN183.

Table 18: In vitro ApoB gene silencing knockdown of liposome formulations in HepG

As shown in Table 18, these formulations showed advantageously high knockdown activity for DX4227 RNAi agents at concentrations of 25 nM and 2.5 nM compared to Qneg.

Example 14

Utilizing liposome formulations prepared from amino acid lipids loaded with peptide condensate carrier particles in vivo Silent knockdown of the ApoB gene

Liposome formulations were prepared using amino acid lipids loaded with peptide condensate carrier particles containing ApoB gene silencing RNAi reagents. The ApoB gene silencing activity of these liposome formulations was determined in mice and compared to mouse serum cholesterol levels. The reduction in ApoB mRNA activity in vivo and the corresponding reduction in serum cholesterol in vivo are shown in Table 19. The liposome formulation in Table 19 was [C18:1-n-Arg-C16/CHEMS/chol/DMPE-PEG2k (50/32/16/2)], and the dose in each case was 2 mg/kg.

Table 19: Silencing of ApoB gene in mice using liposome formulations loaded with peptide condensate carrier particles because

The results in Table 19 show that liposome formulations loaded with peptide condensate carrier particles containing ApoB gene silencing RNAi agents are advantageously well tolerated in mice, because the overall body weight gain at 48 hours after administration was higher than The same formulation without the peptide condensate carrier particles.

In addition, the results in Table 19 also show that the liposome formulation loaded with peptide condensate carrier particles has favorable high activity on ApoB gene silencing in vivo (both in reducing ApoB mRNA and lowering serum cholesterol). The results in Table 19 show that a higher initial N/P of 1.0 and a lower final N/P of 0.6-0.7 are preferred.

Other liposome formulations were prepared using amino acid lipids loaded with carrier particles of peptide condensates containing ApoB gene silencing RNAi agents. The ApoB gene silencing activity of these liposome formulations was determined in mice and compared to mouse serum cholesterol levels. ApoB mRNA lowering activity in vivo and corresponding serum cholesterol lowering in vivo are shown in Table 20.

Table 20: ApoB Gene in Mice Using Liposome Formulations Loaded with Peptide Condensate Carrier Particles silence

For the liposome formulations loaded with peptide condensate carrier particles in Table 20, the delivery efficiency ratio of formulation 3 was 9.21, and that of formulation 4 was 9.86.

The results in Table 20 show that liposome formulations loaded with peptide condensate carrier particles containing ApoB gene silencing RNAi agents are advantageously well tolerated in mice due to body weight gain 48 hours after administration, whereas Body weight was reduced for the same formulation without the peptide condensate carrier particles.

In addition, the results in Table 20 also show that the liposome formulation loaded with peptide condensate carrier particles has favorable high activity on ApoB gene silencing in vivo (both in reducing ApoB mRNA and lowering serum cholesterol).

Claims (34)

1. a method of preparing the compositions that comprises activating agent, described method comprises:

A) provide first fluid, the aqueous buffer solutions that described first fluid comprises activating agent, wherein, described activating agent is selected from gene silencing agent, Gene regulation agent, antisense reagent, peptide nucleic acid(PNA) reagent, ribozyme reagent, RNA reagent or DNA reagent, and medical compounds;

B) provide second fluid, the non-aqueous solution that described second fluid comprises one or more liposomees formation compounds in being easy to the organic solvent mixing with water, alkanol, alkanol-water, acetonitrile, acetone, ketone, dimethyl sulfoxide, surfactant solution, detergent solution and their mixture, wherein, described liposome formation compound is one or more DILA2 amino-acid compounds;

C) described first fluid and described second fluid are clashed into, form thus described organic solvent concentration and be 20% to 50%v/v and pH be 6 to 7.4 shock fluid;

D) in collection container, at the temperature of 20 ℃ to 35 ℃, described in incubation, clash into fluid 0.5 hour to 8 hours, form thus the incubation thing that contains liposome;

Wherein, the volume flow rate of described first fluid is the twice of volume flow rate of described second fluid or higher;

Wherein DILA2 amino-acid compound is suc as formula a series of DILA2 amino-acid compound and the salt thereof shown in I:

R ³-(C=O)-Xaa-Z-R ⁴formula I

Wherein

Xaa has general formula-NR ⁿ-CR ¹r ²-(C=O)-any D-or L-amino acid residue, or the peptide of 2 to 20 amino acid residues, wherein

R ¹be non-hydrogen, replace or unsubstituted amino acid side chain;

R ²hydrogen or by carbon, oxygen, nitrogen, sulfur and hydrogen atom form and have organic group or C (1-5) alkyl of 1 to 20 carbon atom, cycloalkyl, cycloalkyl-alkyl, C (3-5) thiazolinyl, C (3-5) alkynyl, C (1-5) alkanoyl, C (1-5) alkanoyloxy, C (1-5) alkoxyl, C (1-5) alkoxy-C (1-5) alkyl, C (1-5) alkoxy-C (1-5) alkoxyl, C (1-5) alkyl-amino-C (1-5) alkyl-, C (1-5) dialkyl-7-amino-C (1-5) alkyl-, nitro-C (1-5) alkyl, cyano group-C (1-5) alkyl, aryl-C (1-5) alkyl, 4-biphenyl-C (1-5) alkyl, carboxyl or hydroxyl,

R ⁿhydrogen or by carbon, oxygen, nitrogen, sulfur and hydrogen atom form and have organic group or C (1-5) alkyl of 1 to 20 carbon atom, cycloalkyl, cycloalkyl-alkyl, C (3-5) thiazolinyl, C (3-5) alkynyl, C (1-5) alkanoyl, C (1-5) alkanoyloxy, C (1-5) alkoxyl, C (1-5) alkoxy-C (1-5) alkyl, C (1-5) alkoxy-C (1-5) alkoxyl, C (1-5) alkyl-amino-C (1-5) alkyl-, C (1-5) dialkyl-7-amino-C (1-5) alkyl-, nitro-C (1-5) alkyl, cyano group-C (1-5) alkyl, aryl-C (1-5) alkyl, 4-biphenyl-C (1-5) alkyl, carboxyl or hydroxyl,

R ³it is the lipotropy tail derived from naturally occurring or synthetic phospholipid, glycolipid, triacylglycerol, phosphoglyceride, sphingolipid, ceramide, sphingomyelins, cerebroside or ganglioside; Or replacement or unsubstituted C (3-22) alkyl, C (6-12) cycloalkyl, C (6-12) cycloalkyl-C (3-22) alkyl, C (3-22) thiazolinyl, C (3-22) alkynyl, C (3-22) alkoxyl or C (6-12) alkoxy-C (3-22) alkyl; Or the lipotropy tail of other naturally occurring or synthetic lipid arbitrarily, and can comprise steroid;

R ⁴it is the lipotropy tail derived from naturally occurring or synthetic phospholipid, glycolipid, triacylglycerol, phosphoglyceride, sphingolipid, ceramide, sphingomyelins, cerebroside or ganglioside; Or replacement or unsubstituted C (3-22) alkyl, C (6-12) cycloalkyl, C (6-12) cycloalkyl-C (3-22) alkyl, C (3-22) thiazolinyl, C (3-22) alkynyl, C (3-22) alkoxyl or C (6-12) alkoxy-C (3-22) alkyl; Or the lipotropy tail of other naturally occurring or synthetic lipid arbitrarily, and can comprise steroid;

Z is NH, O, S ,-CH ₂s-,-CH ₂s (O)-or by 1 to 40 that is selected from hydrogen, carbon, oxygen, nitrogen and sulphur atom former molecular organic connexon.

2. the method for claim 1, wherein alkanol is C1-C6 alkanol.

3. method as claimed in claim 2, wherein C1-C6 alkanol is ethanol, isopropyl alcohol, isobutanol, sec-butyl alcohol, the tert-butyl alcohol.

4. the method for claim 1, wherein alkanol-water is alcohol-water.

5. the method for claim 1, it further comprises that the buffer agent that is enough to make the concentration of described organic solvent to be less than 20%v/v by adding in described incubation thing quenches described incubation thing.

The method of claim 1, wherein described liposome to form a kind of in compound be PONA, i.e. PONA.

7. the method for claim 1, it further comprises that making the volume flow rate of described first fluid is three times of volume flow rate of described second fluid or higher.

8. the method for claim 1, it further comprises that making the volume flow rate of described first fluid is five times of volume flow rate of described second fluid or higher.

9. the method for claim 1, it further comprises that by the pH regulator of described shock fluid be 3 to 6.

10. the method for claim 1, it is further included in incubation under 3 to 6 pH.

11. the method for claim 1, it further comprises to described collection container and adds buffer agent to regulate the concentration of described organic solvent.

12. the method for claim 1, it further comprises that described activating agent is wrapped in liposome to be greater than 50% level.

13. the method for claim 1, it further comprises that described activating agent is wrapped in liposome to be greater than 70% level.

14. the method for claim 1, wherein described activating agent be UsiRNA.

15. the method for claim 1, wherein the reticent activity of described liposome composition maintainer gene at the temperature of 45 ℃ reach 7 days.

16. the method for claim 1, wherein described liposome composition at the temperature of 45 ℃, keep parcel described activating agent reach 7 days.

17. the method for claim 1, wherein after tangential flow filtration, described liposome size homogeneous, and its diameter is less than 160nm.

18. the method for claim 1, wherein after tangential flow filtration, described liposome size homogeneous, and its average diameter is 40nm to 160nm.

19. the method for claim 1, wherein after tangential flow filtration, described liposome size homogeneous, and its average diameter is 80nm to 150nm.

20. the method for claim 1, it further comprises by tangential flow filtration and diafiltration and filters described incubation thing.

21. the method for claim 1, it further comprises described incubation thing is carried out to sterilizing.

22. the method for claim 1, it further comprises with different pharmaceutically acceptable buffer agents and exchanges described organic solvent.

23. the method for claim 1, it further comprises that to adding in described first fluid organic solvent to make its concentration be 1% to 40%v/v.

24. the method for claim 1, wherein described organic solvent be that C1-C6 alkanol concentration is 40% to 99%v/v Injectable sterile aqueous solution.

25. the method for claim 1, wherein described organic solvent be that C1-C6 alkanol concentration is 70% to 95%v/v Injectable sterile aqueous solution.

26. the method for claim 1, wherein described incubative time be 1 hour to 4 hours.

27. pharmaceutical compositions that in claim 1-26 prepared by the method described in any one.

28. 1 kinds of in vitro method to biological cell delivery of therapeutic nucleic acid, it comprises according to the method described in any one in claim 1-26 prepares compositions and with cell described in described compositions-treated, wherein, described in vitro method is for non-diagnosis and therapeutic purposes.

29. 1 kinds of in vitro method that suppress gene expression in biological cell, it comprises according to the method described in any one in claim 1-26 prepares compositions and with cell described in described compositions-treated, wherein, described in vitro method is for non-diagnosis and therapeutic purposes.

Compositions prepared by the method in 30. claim 1-26 described in any one application in preparing medicine, wherein said medicine is used for suppressing mammiferous gene expression.

The application of 31. compositionss of preparing according to the method described in any one in claim 1-26 in the medicine of preparation treatment disease, wherein said disease comprises cancer, hepatopathy, hypercholesterolemia, diseases associated with inflammation, metabolic disease, fracture, heart disease and viral disease.

32. application according to claim 31, wherein said disease is bladder cancer, hepatocarcinoma, arthritis or encephalitis.

33. application according to claim 32, wherein said arthritis is rheumatoid arthritis.

34. application according to claim 31, wherein said disease is inflammation.